Genome editing using effector oligonucleotides for therapeutic treatment

ABSTRACT

The invention provides compositions and methods of making and using effector oligonucleotides, including effector oligonucleotides with greater than one mismatch as compared to its target sequence. These effector oligonucleotides are useful for improving the efficiency of genomic editing as well as providing therapeutic benefits to individuals in need thereof.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of priority of U.S. provisionalpatent application Ser. No. 61/801,822, filed Mar. 15, 2013 and U.S.provisional patent application Ser. No. 61/867,522, filed Aug. 19, 2013,the contents of which are hereby incorporated by reference herein intheir entirety.

SUBMISSION OF SEQUENCE LISTING ON ASCII TEXT FILE

The content of the following submission on ASCII text file isincorporated herein by reference in its entirety: a computer readableform (CRF) of the Sequence Listing (file name: 873892000140.txt, daterecorded: Mar. 13, 2014, size: 26,630 bytes).

BACKGROUND OF THE INVENTION

Oligonucleotides which are partially complementary to genomic DNAsequence can associate with duplex DNA in a sequence specific manner.See, e.g., WO2011/133802. Oligonucleotides comprising sequences thatvary in part from corresponding genomic DNA sequences have been used toedit chromosomal DNA by recombination of the sequence encoded by theoligonucleotide into the genome.

Whereas oligonucleotides am also used in biological research, a mainintended use of oligonucleotides in cell therapy is to correct geneticsequences associated with disease in cells to produce preparations oftherapeutic cells for use. However, the efficiency ofoligonucleotide-mediated alteration of the genome is very low, limitingthe applicability of this approach. At the same time, due to theinefficiency of the process, it is thought that off-target alteration ofthe genome is also low. Consequently, methods that utilizeoligonucleotides to produce therapeutic cells without increasing theirlow expected off-target activity and that produce increased yields ofoptimally-modified cells would be of interest to enable more effectiveand safe cell therapy.

The sequence of an oligonucleotide used to introduce a geneticalteration is typically designed to correspond to one strand of a targetdouble stranded chromosomal DNA sequence of interest, usually the codingsequence or promoter of a gene of interest wherein the oligonucleotideis additionally designed to comprise a mismatch compared to the targetsequence. Oligonucleotides are thought to associate with theircorresponding target sequences in the genome. In some cases, themismatched sequence is introduced into the genome followingrecombination events.

The efficiency of oligonucleotide-mediated alteration of the genome islow. Various publications teach that oligonucleotides should be designedto contain sequences that are essentially identical to theircorresponding target sequences in order to work. For example, US2010/0172882 teaches that a greater number of homologous positionswithin the oligonucleotide will increase the probability that it will berecombined into the target sequence, target region or target site. Thus,having more mismatches is discouraged by existing publications. Manyoligonucleotides used to alter the genome via recombination reported todate have been designed to introduce a point mutation or to change asingle base. See, e.g., Bonner M., et al. 1:e18. doi:10.1038/mtna.2012.9 Mol. Ther. Nucleic Acids (2012); Bertoni C, et al.37(22):7468-82. doi: 10.1093/nar/gkp757 Nucleic Acids Res. (2009) andAndrieu-Soler C, et al. Nucleic Acids Res. 7; 33(12):3733-42 (2005).

In order to increase the efficiency of genome editing usingoligonucleotides, other references (e.g., US 2011/0262406) teach the useof triplex-forming molecules, which is described as a pair ofsingle-stranded molecules, or a single molecule composed of a pair ofmolecules connected by a linker, that facilitate strand displacement andtriplex formation, in which one molecule binding to the target strand byHoogsteen binding and the other molecule binds to the target strands byWatson-Crick binding in a sequence specific manner. It is thought thatthese molecules recruit cellular factors that are involved inrecombination which work to increase the efficiency with whicholigonucleotides designed to edit the genome are recombined into thegenome, and they have been used in editing the genome of cells for thetreatment of various diseases, such as HIV. The use of multiplecompounds here is one way of approaching genomic editing. Nonetheless,even in combination with these molecules, the efficiency of genomemodification using oligonucleotides remains low. There is a need tosolve the problem of inefficient recombination, inefficient genomicediting, and increased off-target activity, as well as additionalbenefits of providing for compositions and methods of producingtherapeutic cells without increasing their low expected off-targetactivity and increasing yields of optimally-modified cells.

Human immunodeficiency virus (HIV) has infected millions of peopleworldwide. Many efforts have been made to combat HIV infection. Someapproaches focus on small molecules to affect the virus' replicationcycle. Other efforts have focused on the cell therapy side. Since CCR5is known to be a co-receptor that is needed for many HIV isolates,efforts have been made to target CCR5 for HIV therapy. See, e.g., WO2009/06336, US 2005/0220772, and US 2009/0202496.

The reported cure of the Berlin Patient has spurred efforts to createcell therapies based on the interpretation that any disruption of CCR5may underlie a safe and effective cell therapy. One report on a patientstudy referred to as the “Berlin Patient” described how the patient wascured following a bone marrow transplant using bone marrow derived froma Δ32 homozygous individual, or an individual carrying only thisparticular variant of the gene and no wild-type or other variant. See,e.g., Allers K, et al. Blood 117(10): 2791-9 (2011). The A32 variant ofCCR5 represents a disrupted variant of the wild-type gene which occursin a small percentage of North Americans and Europeans but is almostnonexistent in Asians and Africans. In order to infect its target cells,HIV must first dock onto its receptor and co-receptor, CD4 and CCR5,respectively, both of which are expressed on the surface of targetcells. Δ32 homozygous individuals are naturally-resistant to HIV/AIDSand otherwise healthy. Thus, it was thought that disruption of CCR5 perse may be used as the basis of an HIV/AIDS cell therapy. However,various mutations of CCR5 (e.g. introduction of stop codon(s)) have notproven to be viable cell therapy approaches.

Attempts have been made to isolate different types of cells, such asstem cells (e.g., cord blood-derived stem cells and hematopoietic stemcells) that have naturally-occurring Δ32 mutations and to expand theseisolated stem cells with naturally-occurring Δ32 mutation. However,there has been not much success with expansion of populations of stemcells with naturally occurring Δ32 mutations. There exists many problemswith making the appropriate mutations in CCR5 that render the modifiedcell to be less likely to be infected with HIV. As such, one problem tobe solved is the modification of CCR5 in cells that are susceptible toHIV infection such that the resultant cells are less likely to beinfected with HIV. In addition, another problem is the isolation and/orexpansion of stem cells with the precise Δ32 mutations to the extentthat there are enough cells for cell therapy for individual infectedwith HIV, suspected of having HIV infection or at risk of HIV infection.Furthermore, another problem exists with isolating and/or purifying theright population of stem cells with the precise Δ32 deletion in CCR5.

BRIEF SUMMARY OF THE INVENTION

The invention provides for, inter alia, compositions and methods ofmaking and using effector oligonucleotides that are designed to alter anucleic acid sequence (target sequence), such as a genomic targetsequence. The effector oligonucleotides of the invention can be used fortreatment of different conditions and/or diseases where genomic editingof two or more bases can be beneficial. Accordingly, in one aspect, theinvention provides for effector oligonucleotides comprising more thanone mismatch as compared to its target sequence.

In some aspects, the invention provides for effector oligonucleotidescomprising more than one mismatch as compared to its target sequence. Inother aspects, the invention provides for effector oligonucleotidescomprising 2 to 100 or more mismatches, including effectoroligonucleotides comprising mismatches selected from the groupconsisting of: 2 to 50 mismatches, 2 to 40 mismatches, 2 to 30mismatches, 5 to 50 mismatches, 5 to 40 mismatches, 5 to 30 mismatches,10 to 50 mismatches, 10 to 40 mismatches, 10 to 30 mismatches, 15 to 50mismatches, 15 to 40 mismatches, 15 to 30 mismatches, 20 to 50mismatches, 25 to 50 mismatches, 25 to 40 mismatches, 2 to 5 mismatches,6 to 10 mismatches, 11 to 15 mismatches, 16 to 20 mismatches, 21 to 25mismatches, 26 to 31 mismatches, 32 to 40 mismatches, 30 to 50mismatches, and 50 or more mismatches as compared to its targetsequence.

In any of the embodiments above, the target sequence is any chromosomalor genomic sequence, including a sequence that comprises a gene, orportion thereof, or a sequence that encodes an mRNA or protein, orportion thereof. In some embodiments, the target sequence comprises aknown allelic variant or form of the gene or portion thereof,single-nucleotide polymorphism (SNP) form of the gene or portionthereof, a full-length or truncated form of a gene, a mutated form ofthe gene or a portion thereof, or a combination of any of these. In someembodiments, the target sequence corresponds to a variant of a gene, orportion thereof, that is linked or associated with a disease orinfection or susceptibility to a disease or infection.

In any of the embodiments above, the target sequence is a virusreceptor. In any of the embodiments above, the mismatch in the effectoroligonucleotide relates to a sequence to be deleted from a virusreceptor. In any of the embodiments above, the effector oligonucleotidecomprises a sequence that does not match a sequence to be deleted, andthat comprises matches to the target sequence before the sequence to bedeleted and comprises matches to the target sequence after the sequenceto be deleted. In any of the embodiments above, the effectoroligonucleotide has about 10 to 200 matches to the target sequencebefore the sequence to be deleted. In any of the embodiments above, theeffector oligonucleotide has about 10 to 200 matches to the targetsequence after the sequence to be deleted. In any of the embodimentsabove, the effector oligonucleotide has about 10 to 200 matches to thetarget sequence before the site deletion and about 10 to 200 matches tothe target sequence after the site of deletion. In any of theembodiments above, the effector oligonucleotide has about 40 to 200matches to the target sequence before the sequence to be deleted. In anyof the embodiments above, the effector oligonucleotide has about 40 to200 matches to the target sequence after the sequence to be deleted. Inany of the embodiments above, the effector oligonucleotide has about 40to 200 matches to the target sequence before the site deletion and about40 to 200 matches to the target sequence after the site of deletion. Inany of the embodiments above, the effector oligonucleotide has about 10to 40 matches to the target sequence before the sequence to be deleted.In any of the embodiments above, the effector oligonucleotide has about10 to 40 matches to the target sequence after the sequence to bedeleted. In any of the embodiments above, the effector oligonucleotidehas about 10 to 40 matches to the target sequence before the sequence tobe deleted and about 10 to 40 matches to the target sequence after thesequence to be deleted. In any of the embodiments above, the effectoroligonucleotide has about 40 to 200 matches to the target sequencebefore the sequence to be deleted and about 10 to 40 matches to thetarget sequence after the sequence to be deleted. In any of theembodiments above, the effector oligonucleotide has about 10 to 40matches to the target sequence before the sequence to be deleted andabout 40 to 200 matches to the target sequence after the sequence to bedeleted.

In any of the embodiments above, wherein the effector oligonucleotidecomprises a sequence that does not match a sequence to be deleted, andthat comprises matches to the target sequence before the sequence to bedeleted and comprises matches to the target sequence after the sequenceto be deleted, the sequence to be deleted comprises 2 or more bases(i.e. 2 or more mismatches). In some embodiments, the sequence to bedeleted is 10 or more bases long. In some embodiments, the sequence tobe deleted is 20 or more bases long. In some embodiments, the sequenceto be deleted is 40 or more bases. In some embodiments, the sequence tobe deleted consists of 2 to 500 bases. In some embodiments, the sequenceto be deleted consists of 20 to 40 bases.

In any of the embodiments above, the target sequence is an HIV receptor.In any of the embodiments above, the HIV receptor is CCR5. In any of theembodiments above, the mismatch in the effector oligonucleotidecorresponds to a 32 base target sequence that is deleted in a delta-32(Δ32) variant of CCR5, wherein the Δ32 variant is compared to thewild-type sequence of CCR5. In any of the embodiments above, theeffector oligonucleotide comprises a sequence that comprises matches tothe target sequence before the sequence to be deleted and comprisesmatches to the target sequence after the sequence to be deleted. In anyof the embodiments above, the effector oligonucleotide comprises atleast 40 matches to a target sequence before the site of the Δ32deletion in CCR5. In any of the embodiments above, the effectoroligonucleotide comprises at least 40 matches to a target sequence afterthe site of the Δ32 deletion in CCR5. In any of the embodiments above,the effector oligonucleotide is selected from the group consisting of:(a) effector oligonucleotides comprising a sequence that corresponds tothe Δ32 variant of CCR5, (b) effector oligonucleotides comprising asequence that corresponds to the Δ32 variant of CCR5 and thatcorresponds to a coding sequence of the Δ32 variant of CCR5, and (c)effector oligonucleotides that are designed to mimic the Δ32 variant ofCCR5.

In other aspects, the invention provides for compositions comprising anyone of the effector oligonucleotides in any of the embodiments above.

In other aspects, the invention provides for isolated, recombinant cellscomprising a target sequence corresponding to any one of the effectoroligonucleotides in any of the embodiments above. In some embodiments,the isolated, recombinant cells comprise a recombined sequence resultingfrom the contacting of a cell comprising a target sequence with aneffector oligonucleotide targeted to the target sequence. In someembodiments, the isolated, recombinant cells comprise a recombinedsequence resulting from the contacting of the cell comprising a targetsequence with any one of the recombined sequence corresponding to theeffector oligonucleotides in any of the embodiments above

In other aspects, the invention provides for methods of genomic editingex vivo, the method comprising contacting a cell with any one of theeffector oligonucleotides in any of the embodiments above underconditions sufficient for entry of the effector oligonucleotide into thecell and allowing the effector oligonucleotide to edit chromosomal DNAof the cell.

In some aspects, the invention provides for the use of an effectoroligonucleotide to correct, alter or eliminate the sequence associatedwith a disease or infection or susceptibility to the disease orinfection, wherein the effector oligonucleotide is targeted to a variantof a gene or portion thereof, that is linked or associated with adisease or infection or susceptibility to a disease or infection.

In some aspects, the invention provides for a method of treating adisease in a subject comprising administering to the subject in needthereof an effective amount of an effector oligonucleotide. In someembodiments of the method of treating a disease in a subject, theeffector oligonucleotide is targeted to a variant of a gene or portionthereof, that is linked or associated with the disease.

In some aspects, the invention provides for a method of treating aninfection in a subject comprising administering to the subject in needthereof an effective amount of an effector oligonucleotide. In someembodiments of the method of treating an infection in a subject, theeffector oligonucleotide is targeted to a variant of a gene or portionthereof, that is linked or associated with the infection.

In some aspects, the invention provides for a method of preventing adisease or infection in a subject comprising administering to thesubject in need thereof an effective amount of an effectoroligonucleotide. In some embodiments of the method of preventing adisease or infection in a subject, the effector oligonucleotide istargeted to a variant of a gene or portion thereof, that is linked orassociated with susceptibility to the disease or infection.

In some aspects, the invention provides for a method of makingrecombinant cells comprising contacting cells comprising a targetsequence within a gene to be altered with an effector oligonucleotidetargeted to the target sequence and comprising more than one mismatchesas compared to the target sequence; and allowing the effectoroligonucleotide to alter the target sequence in the cells. In someembodiments, the method of making the recombinant cells having aspecific gene alteration comprises: (a) delivering the effectoroligonucleotide targeted to a target sequence and comprising more thanone mismatch as compared to the target sequence into the cellscomprising the target sequence within the gene to be altered; and (b)incubating the cells under conditions sufficient for entry of theeffector oligonucleotide into the cells and allowing the effectoroligonucleotide to alter the target sequence in the cells, to providethe recombinant cells.

In some aspects, the invention provides for a method of making asubstantially enriched population of recombinant cells, the methodcomprising contacting cells comprising a target sequence within a geneto be altered with an effector oligonucleotide targeted to the targetsequence and comprising more than one mismatches as compared to thetarget sequence; allowing the effector oligonucleotide to alter thetarget sequence in the cells; isolating at least one of the recombinantcells; and growing the at least one recombinant cell under conditions toprovide the substantially enriched population of recombinant cells. Insome embodiments, the method of making the substantially enrichedpopulation of recombinant cells comprises: (a) delivering the effectoroligonucleotide targeted to a target sequence and comprising more thanone mismatch as compared to the target sequence into the cellscomprising the target sequence within the gene to be altered; (b)incubating the cells under conditions sufficient for entry of theeffector oligonucleotide into the cells and allowing the effectoroligonucleotide to alter the target sequence in the cells, to providethe recombinant cells; (c) isolating at least one of the recombinantcells; and (d) growing the at least one recombinant cell underconditions to provide the substantially enriched population ofrecombinant cells. In some embodiments, the substantially enrichedpopulation of recombinant cells is further purified by separating therecombinant cells from the growth media and suspending the cells in asuitable media for use in cell therapy.

In some aspects, the invention provides for a method of treating adisease in a subject comprising administering to the subject in needthereof an effective amount of a recombinant cell comprising arecombined sequence resulting from the contacting of a cell comprising atarget sequence within a gene with an effector oligonucleotide targetedto the target sequence and comprising more than one mismatch as comparedto the target sequence under conditions sufficient for entry of theeffector oligonucleotide into the cell and allowing the effectoroligonucleotide to alter the target sequence. In some embodiments of themethod of treating a disease in a subject, the effector oligonucleotideis targeted to a variant of a gene or portion thereof, that is linked orassociated with the disease.

In some aspects, the invention provides for a method of treating aninfection in a subject comprising administering to the subject in needthereof an effective amount of a recombinant cell comprising arecombined sequence resulting from the contacting of a cell comprising atarget sequence within a gene with an effector oligonucleotide targetedto the target sequence and comprising more than one mismatch as comparedto the target sequence under conditions sufficient for entry of theeffector oligonucleotide into the cell and allowing the effectoroligonucleotide to alter the target sequence. In some embodiments of themethod of treating an infection in a subject, the effectoroligonucleotide is targeted to a variant of a gene or portion thereof,that is linked or associated with the infection. In some embodiments,the infection is HIV and the subject has or is suspected of having anHIV infection.

In some aspects, the invention provides for a method of preventing adisease or infection in a subject comprising administering to thesubject in need thereof an effective amount of a recombinant cellcomprising a recombined sequence resulting from the contacting of a cellcomprising a target sequence within a gene with an effectoroligonucleotide targeted to the target sequence and comprising more thanone mismatch as compared to the target sequence under conditionssufficient for entry of the effector oligonucleotide into the cell andallowing the effector oligonucleotide to alter the target sequence. Insome embodiments of the method of preventing a disease or infection in asubject, the effector oligonucleotide is targeted to a variant of a geneor portion thereof, that is linked or associated with susceptibility tothe disease or infection. In some embodiments, the infection is HIV andthe subject is susceptible to infection by HIV.

In any of the embodiments above, the cell is selected from the groupconsisting of mammalian cells, human cells, animal cells, plant cells,yeast cells, insect cells, and reptilian cells. In any of theembodiments above, the mammalian cell is selected from the groupconsisting of embryonic stem cells, induced pluripotent stem cells,adult stem cells, hematopoietic stem cells, cord blood stem cells,cancer stem cells, multipotent progenitor cells, lineage-restrictedprogenitor cells, common myeloid progenitor cells,Granulocyte-macrophage progenitor cells, megakaryocyte-erythroidprogenitor cells, immune cells, differentiated immune cells andCD4-positive immune cells. In any of the embodiments above, the methodfurther comprises contacting the cell with triplex-formingoligonucleotides or pseudocomplementary oligonucleotides. In any of theembodiments above, the triplex-forming oligonucleotide comprises a PNA.In any of the embodiments above, the method further comprises detectionof the cells comprising the sequence encoded by the effectoroligonucleotide by using fluorogenic oligonucleotide probes. In any ofthe embodiments above, the detected cells are isolated byfluorescence-activated cell sorting.

In some aspects, the invention provides for a composition comprising arecombinant cell comprising an altered target sequence and an effectoroligonucleotide targeted to the target sequence. In some embodiments,the effector oligonucleotide is complementary to at least a portion ofthe altered target sequence without any mismatches. In some embodiments,the altered target sequence is the result of a Δ32 deletion of the CCR5gene. In some embodiments, the composition further comprises a detectiontool. In some embodiments, the detection tool is a fluorogenicoligonucleotide probe as used in Chromovert® technology.

In some aspects, the invention provides for, inter alia, compositions ofstem cells and populations of stem cells that have been engineered formodifications in the CCR5 gene and methods of making and using thesepopulations of modified stem cells. These modifications (e.g., Δ32deletion) result in frameshift mutations that render resultant stemcells and populations of stem cells more refractory to HIV infection.

In one aspect, the invention provides for compositions comprising asubstantially pure population of recombinant stem cells, wherein thestem cells comprise a modification in the CCR5 gene that results in aframeshift. In one embodiment the modification is a Δ32 deletion in aCCR5 gene. In other embodiments, the Δ32 mutation comprises deletion ofSEQ ID NO:4 from the wild-type CCR5 gene. In any of the embodiments andcombination of embodiments herein, at least 5, 10, 15, 20, 25, 30, 35,40, 45, 50, 60, 70, 80 or 90% of the stem cells in the populationcomprise the Δ32 deletion. In any of the embodiments and combination ofembodiments herein, at least 50% of the stem cells in the populationcomprise the Δ32 deletion. In any of the embodiments and combination ofembodiments herein, 5 to 90%, 10 to 90%, 15 to 90%, 20 to 90%, 25 to90%, 30 to 90%, 35 to 90%, 40 to 90%, 45 to 90%, or 50 to 90%, of thestem cells in the population comprise the Δ32 deletion. In any of theembodiments and combination of embodiments herein, the stem cell isselected from the group consisting of: embryonic stem cells,induced-pluripotent stem cells, hematopoietic stem cells, adult stemcells, cord blood stem cells, cancer stem cells, multipotent progenitorcells, lineage-restricted progenitor cells, common myeloid progenitorcells, Granulocyte-macrophage progenitor cells andmegakaryocyte-erythroid progenitor cells. In any of the embodiments andcombination of embodiments herein, the stem cells in the populationexpress one or more of the markers selected from the group consistingof: CD34, CD133, CD105, CD45, CD59, Thy1/CD90, C-kit (CD117) and SLAMfamily of cell surface markers. In any of the embodiments andcombination of embodiments herein, at least 5, 10, 15, 20, 25, 30, 35,40, 45, 50, 60, 70, 80 or 90% of the stem cells express one or more ofthe markers selected from the group consisting of: CD34, CD133, CD105,CD45, CD59, Thy1/CD90, C-kit (CD117) and SLAM family of cell surfacemarkers. In any of the embodiments and combination of embodimentsherein, at least 50% of the stem cells express one or more of themarkers selected from the group consisting of: CD34 CD133, CD105, CD45,CD59, Thy1/CD90, C-kit (CD117) and SLAM family of cell surface markers.In any of the embodiments and combination of embodiments herein, 5 to90%, 10 to 90%, 15 to 90%, 20 to 90%, 25 to 90%, 30 to 90%, 35 to 90%,40 to 90%, 45 to 90%, or 50 to 90%, of the stem cells express one ormore of the markers selected from the group consisting of: CD34, CD133,CD105, CD45, CD59, Thy1/CD90, C-kit (CD117) and SLAM family of cellsurface markers. In any of the embodiments and combination ofembodiments herein, the SLAM family of cell surface markers is selectedfrom the group consisting of CD48, CD150, and CD244. In any of theembodiments and combination of embodiments herein, the stem cells in thepopulation do not express one or more of the markers selected from thegroup consisting of: CD13, CD33, CD71, CD19, and CD61. In any of theembodiments and combination of embodiments herein, the stem cells in thepopulation comprise an RNA corresponding to an intracellular,non-cell-surface-localized or a cell-surface localized stem cell marker.In any of the embodiments and combination of embodiments herein, thestem cell marker is selected from the group consisting of: transcriptionfactor gene families, signal pathway genes, and kinase genes. In any ofthe embodiments and combination of embodiments herein, the Δ32 deletionis in one allele. In any of the embodiments and combination ofembodiments herein, the Δ32 deletion is in both alleles. In any of theembodiments and combination of embodiments herein, the stem cells of thepopulation further comprise a detection tool wherein the detection toolallows for detection of a stem cell with Δ32 deletion. In any of theembodiments and combination of embodiments herein, the detection toolcomprises a fluorophore. In any of the embodiments and combination ofembodiments herein, the detection tool further comprises a quencher. Inany of the embodiments and combination of embodiments herein, thedetection tool is a fluorogenic oligonucleotide probe as used inChromovert® technology.

In other aspects, the invention provides methods of making a compositioncomprising a substantially pure population of recombinant stem cells, ora population of cells enriched for recombinant stem cells, wherein thestem cells comprise Δ32 deletion in a CCR5 gene, said method comprising:(a) delivering one or more effector oligonucleotides capable of deletingthe 32 base pair sequence of SEQ ID NO:4 into one or more stem cell(s);(b) culturing the stem cells to increase the number of stem cells,thereby producing a substantially pure population of recombinant stemcells comprising Δ32 deletion. In any of the embodiments and combinationof embodiments herein, the method further comprises introducing adetection tool into the stem cells of step (a). In any of theembodiments and combination of embodiments herein, the detection tool isa fluorogenic oligonucleotide probe as used in Chromovert® technology.

In other aspects, the invention provides methods of treating anindividual with HIV infection or at risk of HIV infection comprisingadministering to the individual an effective amount of any of thecompositions of modified stem cells and/or composition comprising apopulation of modified stem cells disclosed herein. In any of theembodiments and combination of embodiments herein, the compositions ofmodified stem cells and/or composition comprising a population ofmodified stem cells are autologous to the individual.

In other aspects, the invention provides for methods of treating anindividual in need thereof comprising administering a pharmaceuticallyacceptable composition comprising one or more effector oligonucleotidesin any of the embodiments above.

In other aspects, the invention provides for methods of treating anindividual in need thereof comprising administering a pharmaceuticallyacceptable composition comprising the recombinant cells of in any of theembodiments above. In any of the embodiments above, the individual hasor is suspected of having human immunodeficiency virus (HIV) infection.

In any of the embodiments above, the effector oligonucleotide is anon-naturally occurring oligonucleotide.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the coding sequence of the CCR5 gene (SEQ ID NO:1) with the32 bases that are deleted in the Δ32 variant of CCR5 underlined.

FIG. 2 provides the corresponding protein sequences of wild-type CCR5(SEQ ID NO:2), also indicating the sequence maintained in the Δ32variants of CCR5. The amino acids of the wild-type variant of CCR5 thatare preserved in the Δ32 variant are indicated in non-underlined text.As the 32 base deletion of the Δ32 variant of CCR5 introduces aframe-shift mutation, the Δ32 variant lacks the amino acids of thewild-type variant of CCR5 indicated in underlined text.

FIG. 3 depicts the Δ32 CCR5 protein sequence (SEQ ID NO:3) with thenovel 31 amino acid C-terminal tail resulting from the Δ32 deletionunderlined.

FIG. 4A depicts effector oligonucleotides used to introduce genomicalterations of greater that one base. Chemical modifications used, ifany, are listed. All of these oligos correspond to the sequence of theΔ32 variant of the CCR5 gene. All oligos were synthesized by Genelink,Inc. and gel purified. Note that the letters ‘T’ or “B” in the oligoname refers to whether the sequence of the oligo corresponds to thesense or antisense strand, respectively. Each oligo name contains twonumbers separated by a period. The first number indicates the number ofbases in the oligo that match the sequence of the CCR5 gene prior to thesite of the 32 base deletion and the second number indicates the numberof bases in the oligo that match the sequence of the CCR5 gene followingthe site of the 32 base deletion present in the Δ32 variant of the CCR5gene.

FIG. 4B shows the alignment of the effector oligonucleotides againsttheir corresponding genomic target. FIG. 4B depicts the position of eacheffector oligonucleotide relative to the sequence of the Δ32 variant ofCCR5. The two long horizontal lines indicate a portion of the CCR5sequence. The thick vertical line indicates the site of the 32 basedeletion that characterizes the Δ32 versus the wild-type variant of theCCR5 gene. The distance between every two vertical lines indicates 10bases. The position of each effector oligonucleotide relative to thissequence is indicated by a horizontal line for each effectoroligonucleotide, which is identified using its numerical identifier.Effector oligonucleotides 1, 3, 5, 7 and 19 correspond in sequence tothe sense or coding strand, whereas effector oligonucleotides 2, 4, 6, 8and 20 correspond in sequence to the non-coding strand.

FIG. 5 depicts the genomic PCR results showing detection of effectoroligonucleotide-mediated alteration of the genome using effectoroligonucleotides to introduce genomic alterations of greater than onebase. K562 cells and THP cells were electroporated with the effectoroligonucleotides as indicated above each lane by the numbering providedin FIG. 1A i.e. as 1, 2, 3, 4, 5, 6, 7, 8, 19, 20 or “C” (controlwithout effector oligonucleotide). K562 and THP cells were eachelectroporated at a high (1,000,000 cells/˜120 μl) or low (500,000cells/˜120 μl) cell density. The top row shows results for K562 cells atlow (columns 1-12) and high (columns 14-23) cell densities. The bottomrow shows results for THP cells electroporated at high (columns 1-12)and at low (columns 14-23) cell densities. “M” indicates columns loadedwith molecular weight marker (column 13, top and bottom rows). Thegenomic PCR was performed on genomic DNA obtained from the cellsapproximately 2 days following electroporation using CCR5-specificgenomic primers. The small gel at the bottom of the figure shows PCRresults using plasmid containing wild-type CCR5 (lane marked W) orplasmid comprising the Δ32 CCR5 mutation (lane marked D) as template,and marker in the lane marked M. The molecular weight marker comprisesDNA bands of 100, 200 and 300 bases, starting from the bottom.

FIG. 6 depicts a schematic of the amino acid (AA) sequences of thewild-type (WT) and Δ32 variants of CCR5. The total number of amino acidsfor each variant is indicated.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides compositions and methods of making and usingeffector oligonucleotides, including effector oligonucleotides withgreater than one mismatch as compared to its target sequence. Theseeffector oligonucleotides are useful for improving the efficiency ofgenomic editing as well as providing therapeutic benefits to individualsin need thereof. The invention also provides compositions of recombinantcells and populations of recombinant cells that have been engineered formodifications in gene sequences using the effector oligonucleotides asdescribed herein.

The invention also provides for, inter alia, compositions of recombinantcells and populations of recombinant cells that have been engineered formodifications in a gene. In some embodiments, the recombinant cells arestem cells engineered for modifications in the CCR5 gene. In someembodiments, these modifications (e.g., Δ32 deletion) result in aframeshift mutation that render the stem cells and cells differentiatedfrom these stem cells (and any intermediate cells in between) lesslikely to become infected by HIV. These recombinant stem cells areuseful for treating individuals with HIV infection, suspected of havingHIV infection, and/or at risk of HIV infection. The invention alsoprovides for methods for making and using these recombinant stem cellsfor cell therapy. The recombinant stem cells can differentiate,transdifferentiate or de-differentiate during the course of thetreatment but the common trait is that the stem cells contain themodifications to CCR5 (e.g, Δ32 mutation) described herin such that thestem cells (and any intermediate and resultant cell population) are morerefractory to HIV infection.

A. General Techniques

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of stem cell biology, cell culturing,molecular biology (including recombinant techniques), microbiology, cellbiology, biochemistry and immunology, which are within the skill of theart. Such techniques are explained fully in the literature, such as,Molecular Cloning: A Laboratory Manual, third edition (Sambrook et al.,2001) Cold Spring Harbor Press; Oligonucleotide Synthesis (Herdewijn,ed., 2004); Animal Cell Culture (R. I. Freshney, ed., 1987); Methods inEnzymology (Academic Press, Inc.; Handbook of Experimental Immunology(D. M. Weir & C.C. Blackwell, eds.); Gene Transfer Vectors for MammalianCells (J. M. Miller & M. P. Calos, eds., 1987); Current Protocols inMolecular Biology (F. M. Ausubel et al., eds., 1987); PCR: ThePolymerase Chain Reaction, (Mullis et al., eds., 1994); CurrentProtocols in Immunology (J. E. Coligan et al., eds., 1991); TheImmunoassay Handbook (D. Wild, ed., Stockton Press NY, 1994);Bioconjugate Techniques (Greg T. Hermanson, ed., Academic Press, 1996);and Methods of Immunological Analysis (R. Masseyeff, W. H. Albert, andN. A. Staines, eds., Weinheim: VCH Verlags gesellschaft mbH, 1993);Short Protocols in Molecular Biology (Wiley and Sons, 1999), EmbryonicStem Cells: A Practical Approach (Notaranni et al. eds., OxfordUniversity Press 2006); Essentials of Stem Cell Biology (R. Lanza, ed.,Elsevier Academic Press 2006); Stem Cell Assays (Methods in MolecularBiology) (Mohan C. Vemuri, Ed., Humana Press; first edition (Aug. 10,2007); Mesenchymal Stem Cells: Methods and Protocols (Methods inMolecular Biology) (Darwin J. Prockop, Donald G. Phinney, Bruce A.Bunnell, Eds., first edition (Mar. 7, 2008)); Handbook of Stem Cells(Robert Lanza, et al., Eds., Academic Press (Sep. 14, 2004); Stem CellCulture Vol 86: Methods in Cell Biology (Jennie P. Mather, Ed., AcademicPress, first edition (May 15, 2008)); Practical Hematopoietic Stem CellTransplantation (Andrew J. Cant, et al. Eds., Wiley-Blackwell, firstedition (Jan. 22, 2007)); Hematopoietic Stem Cell Protocols (Kevin D.Bunting, Ed., Humana Press, 2nd ed. edition (Jan. 31, 2008)); BoneMarrow and Stem Cell Transplantation (Methods in Molecular Medicine)(Meral Beksac, Ed., Humana Press; first edition (May 3, 2007)); StemCell Therapy and Tissue Engineering for Cardiovascular Repair: FromBasic Research to Clinical Applications (Nabil Dib, et al., Eds.,Springer, first edition (Nov. 16, 2005)); Blood And Marrow Stem CellTransplantation: Principles, Practice, And Nursing Insights (KimSchmit-Pokorny (Author) and Susan Ezzone (Editor), Jones & BartlettPublishers; third edition (May 22, 2006)); Hematopoietic Stem CellProtocols (Christopher A. Klug and Craig T. Jordan, Eds., Humana Press;first edition (Dec. 15, 2001)); and Clinical Bone Marrow and Blood StemCell Transplantation (Kerry Atkinson, et al., Eds., Cambridge UniversityPress; third edition (Dec. 8, 2003)).

B. Definitions

As used herein, the singular form “a”, “an”, and “the” includes pluralreferences unless indicated otherwise. For example, “an” effectoroligonucleotide includes one or more Effector oligonucleotides.

Reference to “about” a value or parameter herein refers to the usualerror range for the respective value readily known to the skilled personin this technical field. Reference to “about” a value or parameterherein includes (and describes) aspects that are directed to that valueor parameter per se. For example, description referring to “about X”includes description of “X.”

It is understood that aspects of the invention described herein include“comprising,” “consisting,” and “consisting essentially of” aspects.

As used interchangeably herein, the terms “polynucleotides” or“oligonucleotide” or “oligo” include single-stranded DNA (ssDNA),double-stranded DNA (dsDNA), single-stranded RNA (ssRNA) anddouble-stranded RNA (dsRNA), modified oligonucleotides andoligonucleosides or combinations thereof. The oligonucleotide can belinearly or circularly configured, or the oligonucleotide can containboth linear and circular segments. Oligonucleotides are polymers ofnucleosides joined, generally, through phosphodiester linkages, althoughalternate linkages, such as phosphorothioate esters may also be used inoligonucleotides. A nucleoside consists of a purine (adenine (A) orguanine (G) or derivative thereof) or pyrimidine (thymine (T), cytosine(C) or uracil (U), or derivative thereof) base bonded to a sugar. Thefour nucleoside units (or bases) in DNA are called deoxyadenosine,deoxyguanosine, deoxythymidine, and deoxycytidine. A nucleotide is aphosphate ester of a nucleoside. Oligonucleotides can also includenon-naturally occurring bases (e.g. modified or substituted bases).Oligonuculeotides may additionally comprise one or more covalentlyattached or linked compounds, toxins, proteins, enzymes, hormones,signaling molecules, fluorescent molecules, quenchers, chemicals andlinkers or a combination thereof. Oligonucleotides may comprise one ormore portions or bases that are covalently crosslinked or attached toother portions or bases.

A “non-naturally occurring oligonucleotide” as used herein refers to anoligonucleotide having at least one modification or substitution in theinternuceloside linkage, sugar moiety or the nucleoside base not foundin natural nucleic acids. The effector oligonucleotides as describedherein include non-naturally occurring oligonucleotides, as furtherdescribed herein.

A “target sequence” as used herein generally refers to any nucleic acidsequence, such as a genomic sequences, that is to be altered by theeffector oligonucleotide, e.g. by contacting a cell with the effectoroligonucleotide under conditions suitable to alter the genomic sequencein the cell. The target sequence can be a sequence within a gene ofinterest, i.e. a gene to be altered, such that the gene of interest canbe altered by altering the target sequence. The effector oligonucleotidematches, or is complementary to, at least a portion of the targetsequence, and also includes at least a portion that does not match thetarget sequence, or wherein the target sequence includes a portion thatdoes not match the effector oligonucleotide (e.g. having at least 2 ormore mismatches).

A “mismatch” or “mismatches” as used herein refers to portions of aneffector oligonucleotide that do not match portions of a targetsequence, or portions of the target sequence that do not match theeffector oligonucleotide, as described herein. The mismatches can beinsertions (e.g. additional bases in the effector oligonucleotide notmatching the target sequence, resulting in addition of bases to thetarget sequence), deletions (e.g. bases missing from the effectoroligonucleotide that are in the target sequence, e.g. wherein theeffector oligonucleotide has portions matching a portion of the targetsequence prior to and a portion of the sequence after a sequence that isin the target sequence that does not match the effector oligonucleotide,resulting in deletion of the unmatched sequence from the targetsequence), or non-complementary bases (e.g. resulting in alterations inthe target sequence), or any combinations thereof. The effectoroligonucleotide is designed to result in altering the target sequence,for example where the resulting altered target sequence matches theeffector oligonucleotide without any mismatches.

An “individual,” “subject” or “patient” is a vertebrate. In certainembodiments, the vertebrate is a mammal. Mammals include, but are notlimited to, primates (including human and non-human primates), pets(e.g., dogs, cats, rabbits, etc.), agricultural animals (e.g., cows,livestock, etc.), sport animals (e.g., horses), and rodents (e.g., miceand rats). In certain embodiments, a mammal is a human.

An “effective amount” or a “sufficient amount” of a substance is thatamount sufficient to effect beneficial or desired results, includingclinical results, and, as such, an “effective amount” depends upon thecontext in which it is being applied. In the context of administering acomposition that comprises one or more effector oligonucleotides of theinvention, an effective amount of an effector oligonucleotide is anamount sufficient to achieve recombination of the effectoroligonucleotide at its corresponding target site in the genome ascompared to recombination of the effector oligonucleotide at other sitesin the genome, or to achieve a greater rate or frequency ofrecombination of the effector oligonucleotide at its correspondingtarget site in the genome as compared to the rate or frequency ofrecombination of the effector oligonucleotide at other sites in thegenome in cells treated with the effector oligonucleotide. When used inthe treatment or prophylaxis context, or in the context of palliatingpain or alleviating the symptoms of a particular condition, an effectiveamount, e.g. of an effector oligonucleotide, or of a recombinant cellresulting from alteration mediated by an effector oligonucleotide, is anamount sufficient to effect beneficial or desired results, includingclinical results. An effective amount can be administered in one or moreadministrations. For purposes of this invention, for example, aneffective amount of recombinant cells (e.g. Δ32 CCR5 stem cells) is acertain amount of the modified cells that can reduce one of moresymptoms of the conditions for which the individual is being treated(e.g., HIV viral burden). As an example, an effective amount of modifiedstem cells (e.g. Δ32 CCR5 stem cells) encompasses the use of thesemodified stem cells when they are being grown or proliferated in theirpluripotent, undifferentiated state as well as the use of modified stemcells when they have been cultured further to induce them todifferentiate down a particular pathway. When used in the context of“assisting therapy,” an effective amount enhances a therapeutic regimen(as compared to a regimen lacking the modified stem cells) and, as such,provides a beneficial or desired result.

As used herein, and as well-understood in the art, “treatment” or“treating” is an approach for obtaining beneficial or desired results,including clinical results. For purposes of this invention, beneficialor desired clinical results include, but are not limited to, alleviationor amelioration of one or more symptoms, diminishment of extent ofdisease, stabilized (i.e., not worsening) state of disease, preventingspread of disease, delay or slowing of disease progression, ameliorationor palliation of the disease state, and remission (whether partial ortotal), whether detectable or undetectable. “Treatment” or “treating”can also mean prolonging survival as compared to expected survival ifnot receiving treatment. “Ireatment” or “treating” can also meanconferring resistance to infection. “Receiving treatment” includesinitial treatment and/or continuing treatment. In some aspects,treatment with a one or more cells disclosed herein is accompanied by noor fewer side effects than are associated with currently availabletherapies. In the context of this invention, the symptoms of AIDS andany AIDS-related conditions are encompassed within this scope.

“Palliating” a disease or disorder means that the extent and/orundesirable clinical manifestations of a disorder or a disease state arelessened and/or time course of the progression is slowed or lengthened,as compared to not treating the disorder. Especially in the case ofHIV/AIDS, as is well understood by those skilled in the art, this caninclude one or more of the following: reducing viral load, or theconcentration or count of virus, in the individual or in the bodilyfuilds of the individual, increasing the number of CD4-positive immunecells in the individual, increasing the strength of immune system, theefficiency or reliability of the immune system or the immune response inindividuals, decreasing or inhibiting the replication or propagation ofHIV in the individual, decreasing or eliminating the integration of thegenetic material corresponding to HIV into the genome of the cells ofthe individual, decreasing or rendering inhospitable or uninfectable thepotential cellular targets or reservoirs in the individual that may beinfected by HIV or used by HIV to replicate or propagate, eliminatingfrom the individual the cells in the individual that are infected orinfectable by HIV or in which the genetic material of HIV has becomeintegrated, mitigating or decreasing the side-effects or negativeeffects of HIV/AIDS on the tissues or organs of the individual, and/ordecreasing the infectiousness of the individual or the propensity orability of the individual to infect others via infected bodily fluids.Further, palliation does not necessarily occur by administration of onedose, but often occurs upon administration of a series of doses of atreatment or therapy. Thus, an amount of a treatment or therapysufficient to palliate a response or disorder may be administered in oneor more administrations.

As used herein, “refractory” or “resistant” to HIV infection refers tothe reduction of (or lessening of) HIV infection in cells that aresusceptible to HIV infection. It can be a lessening of the rate ofinfection, lessening the viral burden of cells infected, reducing theviral burden in a biological sample being tested as well as at thecellular level. It is not intended to require that 100% of the cells ina particular population or sample are not infected.

As used herein, “delaying” development of a disease or condition meansto defer, hinder, slow, retard, stabilize and/or postpone development ofthe disease or condition. This delay can be of varying lengths of time,depending on the history of the disease and/or individual being treated.As is evident to one skilled in the art, a sufficient or significantdelay can, in effect, encompass prevention, in that the individual doesnot develop the disease or condition. For example, the method may reducethe probability of disease development in a given time frame and/orreduce the extent of the disease in a given time frame, when compared tonot using the method. In some aspects, such comparisons are based onclinical studies using a statistically significant number ofindividuals. Disease development can be detectable using standardclinical techniques. Development may also refer to disease progressionthat can be initially undetectable and includes occurrence, recurrence,and onset.

As used herein, “in need thereof” includes individuals who have acondition or disease or are “at risk” for the condition or disease. Asused herein, an “at risk” individual is an individual who is at risk ofdevelopment of a condition. An individual “at risk” may or may not havea detectable disease or condition, and may or may not have displayeddetectable disease prior to the treatment methods described herein. “Atrisk” denotes that an individual has one or more so-called risk factors,which are measurable parameters that correlate with development of adisease or condition and are known in the art. An individual having oneor more of these risk factors has a higher probability of developing thedisease or condition than an individual without these risk factor(s).These risk factors include, but are not limited to, age, sex, diet,history of previous disease, presence of precursor disease, genetic(i.e., hereditary) considerations, breeding protocols andconsiderations, and environmental exposure.

“Stem cells” as used herein refers to cells with self-regenerationcapacities and the ability to become another type of cell in itsdifferentiation pathway. As used herein, stem cells are cells that aretotipotent, pluripotent, multipotent, oligopotent and/or unipotent.

As used herein, “delta-32” and “Δ32” are interchangeable and refers to astretch of 32 bases in the CCR5 sequence that is removed from thesequence of CCR5 (see FIG. 1).

As used herein, the term “isolated population” of stem cells refers to apopulation of one or more stem cells that have been manipulated toprovide a preparation of cells that is substantially free of additionalcomponents (e.g., cellular debris). Various aspects of isolatedpopulations are described herein. The term “isolated population” of stemcells can also refer to a population of one or more stem cells that havebeen manipulated or enriched for cells that have been manipulated,including genetically manipulated, that have been isolated or enrichedfrom a population of cells that were treated to manipulate them. Forexample, some population of cells that are treated to form the alteredcells will not be altered, and the altered cell population can beisolated or enriched relative to the unaltered population.

As used herein, the term “homogeneous population” and “highlyhomogeneous population” and “enriched population” of stem cells refer toa population of cells where a significant portion of the population isstem cells, or where a population of stem cells is altered to provide arecombinant stem cell which can be enriched relative to the unalteredstem cells, or where a significant portion of the population is stemcells having a specific genotype with respect to one or more sequencesor markers. Various embodiments reflecting homogeneity including degreesof homogeneity are described herein and can also be described using theterm “substantially pure” or “substantially enriched”.

“Purity” as used to describe the purity of stem cells does notnecessarily refer to the presence of only stem cells in the compositionbut rather indicates that the stem cells have been manipulated such thatthey have been removed from their natural tissue environment andindicates their relationship to the other cells present in the resultingpopulation. “Purity” as used to describe the purity of stem cells canalso indicate that the stem cells have been manipulated such that theyhave a specific genotype with respect to one or more sequences ormarkers.

By “pharmaceutically acceptable carrier” is meant any material which,when combined with an active ingredient, allows the ingredient to retainbiological activity and does not provoke an unacceptable immune response(e.g., a severe allergy or anaphylactic shock) based on the knowledge ofa skilled practitioner. Examples include, but are not limited to, any ofthe standard pharmaceutical carriers such as carboxymethylcellulose(CMC), phosphate buffered saline solutions, water, emulsions such asoil/water emulsion, and various types of wetting agents. Exemplarydiluents for aerosol or parenteral administration are phosphate bufferedsaline or normal (0.9%) saline. An exemplary carrier for the infusion ofcells is CMC. Compositions comprising such carriers are formulated bywell known conventional methods (see, for example, Remington'sPharmaeutical Sciences, 18th edition, A. Gennaro, ed., Mack PublishingCo., Easton, Pa., 1990; and Remington, The Science and Practice ofPharmacy 20th Ed. Mack Publishing, 2000, which are each herebyincorporated by reference in their entireties, particularly with respectto formulations).

General reference to “the composition” or “compositions” includes and isapplicable to compositions of the invention. The invention also providespharmaceutical compositions comprising the components described herein.

C. Effector Oligonucleotides

Effector oligonucleotides are oligonucleotides of varying length thatcan bind to target sequences. Different types of target sequences,including those genes with causal or correlative relationship todiseases and disease states are contemplated within the scope of theinvention. Different types of target sequences, including those geneswith causal or correlative relationship to infections or susceptibilityto infection by infectious agents or viruses are contemplated within thescope of the invention. Target sequences are described in greater detailbelow.

In one aspect, the effector oligonucleotide has one mismatch as comparedto its target sequence. In other aspects, the effector oligonucleotidehas more than one mismatch as compared to its target sequence. Theseeffector oligonucleotides with mismatch(es) can be used to producegreater alterations of the genome, for instance to add, substitute ordelete more than one DNA base from a target sequence. Such genomicalterations of the genome could be used for instance to add, alter ordelete amino acids to an encoded protein, modify a promoter sequence andits activity, modify a splice site and alternative splicing, modifyother gene-regulatory elements or DNA binding sites within the genome.Accordingly, effector oligonucleotides of the invention can have morethan one mismatch, such as 2 to 500 mismatches, for example, greaterthan 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 to 15, 16 to 25, 26 to 40, 41 to 100or great than 100 mismatches. In some embodiments, the effectoroligonucleotide has 2 to 500, 2 to 400, 2 to 300, 2 to 200, 2 to 100, 5to 500, 5 to 400, 5 to 300, 5 to 200, 5 to 100, 10 to 500, 10 to 400, 10to 300, 10 to 200, 10 to 100, 15 to 500, 15 to 400, 15 to 300, 15 to200, 15 to 100, 20 to 500, 20 to 400, 20 to 300, 20 to 200, 20 to 100,25 to 500, 25 to 400, 25 to 300, 25 to 200, 25 to 100, 30 to 500, 30 to400, 30 to 300, 30 to 200, 30 to 100, 35 to 500, 35 to 400, 35 to 300,35 to 200, 35 to 100, 40 to 500, 40 to 400, 40 to 300, 40 to 200, 40 to100, 45 to 500, 45 to 400, 45 to 300, 45 to 200, 45 to 100, 50 to 500,50 to 400, 50 to 300, 50 to 200, 50 to 100, 2 to 75, 2 to 50, 2 to 40, 2to 30, 5 to 75, 5 to 50, 5 to 40, 5 to 30, 10 to 75, 10 to 50, 10 to 40,10 to 30, 15 to 75, 15 to 50, 15 to 40, 15 to 30, 20 to 75, 20 to 50, 20to 40, 20 to 30, 25 to 75, 25 to 50, 25 to 40, 25 to 30, 30 to 75, 30 to50, 30 to 40, 2 to 5, 6 to 10, 11 to 15, 16 to 20, 21 to 25, 26 to 31,32 to 40, 40 to 50, or 50 or more mismatches as compared to its targetsequence.

Effector oligonucleotides that can be used to alter the genome by morethan one DNA base are described. These effector oligonucleotides can besynthesized to have varying lengths, including 20 to 10000 bases, forinstance 20, 20-500, 20-400, 20-300, 20-200, 20-100, 21-30, 31-40,41-50, 51-60, 61-70, 71-80, 81-90, 91-100, 100-500, 100-400, 100-300,100-200, 101-120, 121-140, 141-160, 161-180, 181-200, 200-500, 200-400,200-300, 201-250, 251-300, 300-500, 300-400, 301-350, 351-400, 401-500,500-600, 600-700, 700-800, 800-900, 900-1000, 1000-1500, 1500-2000,2000-3000, 3000-4000, 4000-5000, 5000-10000, or greater than 10000bases. The effector oligonucleotides may comprise one or more sites thatcomprise a mismatch to their corresponding target sequences, where atleast one of these sites comprises a mismatch that is 2-100 mismatchedbases, such as at least of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 20-25, 25-30, 31, 32, 33, 34, 35, 35-40, 40-50,50-60, 60-70, 70-80, 80-90, 90-100, or greater than 100 mismatchedbases. Where two or more sites within an effector oligonucleotidecomprise mismatched bases, the two or more sites may each be spacedapart from another by 1-100 bases, such as 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 20-25, 25-30, 31, 32, 33,34, 35, 35-40, 40-50, 50-60, 60-70, 70-80, 80-90, 90-100 or greater than100 bases.

Effector oligonucleotides can be used to alter the genome by deletion ofa sequence from a gene. In some embodiments, such effectoroligonucleotides comprise a sequence that matches a first portion of atarget sequence and a second portion of a target sequence, wherein thefirst portion of the target sequence is before the sequence to bedeleted and the second portion of the target sequence is after thesequence to be deleted, wherein the effector oligonucleotide does notmatch the sequence to be deleted. In some embodiments, the effectoroligonucleotide comprises the sequence that matches the first portion ofthe target sequence directly linked to a sequence that matches thesecond portion of the target sequence. In some embodiments, the sequencethat matches the target sequence before the sequence to be deleted is 10to 500, 10 to 400, 10 to 300, 10 to 200, 10 to 125, 10 to 60, 20 to 500,20 to 400, 20 to 300, 20 to 200, 20 to 125, 20 to 60, 30 to 500, 30 to400, 30 to 300, 30 to 200, 30 to 125, 30 to 60, 40 to 500, 40 to 400, 40to 300, 40 to 200, 40 to 125, or 40 to 60 bases. In some embodiments,the sequence that matches the target sequence after the sequence to bedeleted is 10 to 500, 10 to 400, 10 to 300, 10 to 200, 10 to 125, 10 to60, 20 to 500, 20 to 400, 20 to 300, 20 to 200, 20 to 125, 20 to 60, 30to 500, 30 to 400, 30 to 300, 30 to 200, 30 to 125, 30 to 60, 40 to 500,40 to 400, 40 to 300, 40 to 200, 40 to 125, or 40 to 60 bases. In someembodiments, the sequence that matches the target sequence before thesequence to be deleted is 10 to 500, 10 to 400, 10 to 300, 10 to 200, 10to 125, 10 to 60, 20 to 500, 20 to 400, 20 to 300, 20 to 200, 20 to 125,20 to 60, 30 to 500, 30 to 400, 30 to 300, 30 to 200, 30 to 125, 30 to60, 40 to 500, 40 to 400, 40 to 300, 40 to 200, 40 to 125, or 40 to 60bases and the sequence that matches the target sequence after thesequence to be deleted is 10 to 500, 10 to 400, 10 to 300, 10 to 200, 10to 125, 10 to 60, 20 to 500, 20 to 400, 20 to 300, 20 to 200, 20 to 125,20 to 60, 30 to 500, 30 to 400, 30 to 300, 30 to 200, 30 to 125, 30 to60, 40 to 500, 40 to 400, 40 to 300, 40 to 200, 40 to 125, or 40 to 60bases. In some embodiments, the sequence to be deleted from the gene is2 to 500, 2 to 400, 2 to 300, 2 to 200, 2 to 100, 5 to 500, 5 to 400, 5to 300, 5 to 200, 5 to 100, 10 to 500, 10 to 400, 10 to 300, 10 to 200,10 to 100, 15 to 500, 15 to 400, 15 to 300, 15 to 200, 15 to 100, 20 to500, 20 to 400, 20 to 300, 20 to 200, 20 to 100, 25 to 500, 25 to 400,25 to 300, 25 to 200, 25 to 100, 30 to 500, 30 to 400, 30 to 300, 30 to200, 30 to 100, 35 to 500, 35 to 400, 35 to 300, 35 to 200, 35 to 100,40 to 500, 40 to 400, 40 to 300, 40 to 200, 40 to 100, 45 to 500, 45 to400, 45 to 300, 45 to 200, 45 to 100, 50 to 500, 50 to 400, 50 to 300,50 to 200, 50 to 100, 2 to 75, 2 to 50, 2 to 40, 2 to 30, 5 to 75, 5 to50, 5 to 40, 5 to 30, 10 to 75, 10 to 50, 10 to 40, 10 to 30, 15 to 75,15 to 50, 15 to 40, 15 to 30, 20 to 75, 20 to 50, 20 to 40, 20 to 30, 25to 75, 25 to 50, 25 to 40, 25 to 30, 30 to 75, 30 to 50, 30 to 40, 2 to5, 6 to 10, 11 to 15, 16 to 20, 21 to 25, 26 to 31, 32 to 40, 40 to 50,or 50 or more bases. In some embodiments, the sequence to be deleted is1-100 bases (i.e. 1-100 bases not matched to the effectoroligonucleotide), 1-200 bases, 1-300 bases, 1-400 bases, or 1-500 bases,such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 1-90, 1-80, 1-70, 1-60, 1-50, 20-25, 25-30, 31, 32, 33, 34, 35,35-40, 40-50, 50-60, 60-70, 70-80, 80-90, 90-100, or greater than 100bases representing a sequence to be deleted from the gene.

Effector oligonucleotides can be produced using DNA bases or chemicallymodified DNA bases. Chemical modifications can include modifications ofDNA bases, sugars, and linkages between these, or any combination ofthese, to provide a non-naturally occurring effector oligonucleotide.Effector oligonucleotides can comprise one or more different kinds ofchemical modifications. In some embodiments, phosphorothioate linkagescan be used. Numerous modifications are known in the art and may all beconsidered for use. In particular, modifications used in siRNAs orantisense oligonucleotides would be of interest, as well as chemicalmodifications that increase the strength of oligonucleotide binding orhybridization to target sequences, specificity of oligonucleotidehybridization to target sequences, efficiency and/or consistency ofoligonucleotide delivery into cells, and/or resistance, stability orhalf-life of the oligonucleotides to degradation in buffers and incells. Non-limiting examples of chemical modifications that may be usedinclude: one or more of the sugar-phosphodiester type backbone, 2′OH,base can be modified. The substitution of the phosphodiester linkageincludes but is not limited to —OP(OH)(O)O—, —OP(O⁻M⁺)(O)O—,—OP(SH)(O)O—, —OP(S⁻M⁺)(O)O—, —NHP(O)₂O—, —OC(O)₂O—, —OCH₂C(O)₂NH—,—OCHC(O)₂O—, —OP(CH₃)(O)O—, —OP(CH₂C₆H₅)(O)O—, —P(S)(O)O— and—OC(O)₂NH—. M⁺ is an inorganic or organic cation. The backbone can alsobe peptide nucleic acid (PNA), where the deoxyribose phosphate backboneis replaced by a pseudo peptide backbone. Peptide nucleic acid isdescribed by Hyrup and Nielsen, Bioorganic & Medicinal Chemistry 4:5-23,1996, and Hydig-Hielsen and Godskesen, WO 95/32305, each of which ishereby incorporated by reference herein.

The 2′ position of the sugar includes but is not limited to H, OH, C₁-C₄alkoxy, OCH₂—CH═CH₂, OCH₂—CH═CH—CH₃, OCH₂—CH═CH—(CH₂).CH₃ (n=0, 1 . . .30), halogen (F, Cl, Br, I), C₁-C₆ alkyl and OCH₃. C₁-C₄ alkoxy andC₁-C₆ alkyl may be or may include groups which are straight-chain,branched, or cyclic.

The oligonucleotide may also include one or more bridged nucleotides(BNA), i.e. nucleic acids having at least one sugar having a bridgebetween two of the sugar ring positions, such as 1′, 4′ or 2′, 4′ or 3′,4′ bridged sugar moieties. See, for example, Takeshi Imanishi andSatoshi Obika, Chem. Commun., 2002, 1653-1659; Mitsuoka et al., NucleicAcids Research, 2009, 37(4):1225-1238; Hari et al., Tetrahedron, 2003,59:5123-5128; Rahman et al., Angew. Chem. Int. Ed., 2007, 46:4306-4309;Rahman et al., J. Am. Chem. Soc., 2008, 130:4886-4896.

The bases of the nucleotide can be any one of adenine, guanine,cytosine, thymine, uracil, inosine, or the forgoing with modifications.Modified bases include but are not limited to N4-methyl deoxyguanosine,deaza or aza purines and pyrimidines. Ring nitrogens such as the N1 ofadenine, N7 of guanine, N3 of cytosine can be alkylated. The pyrimidinebases can be substituted at position 5 or 6, and the purine bases can besubstituted at position 2, 6 or 8. See, for example, Cook, WO 93/13121;Sanger, Principles of Nucleic Acid Structure, Springer-Verlag, New York(1984), incorporated herein by reference.

Derivatives of the conventional nucleotide are well known in the art andinclude, for example, molecules having a different type of sugar. The04′ position of the sugar can be substituted with S or CH₂. For example,a nucleotide base recognition sequence can have cyclobutyl moietiesconnected by linking moieties, where the cyclobutyl moieties havehetereocyclic bases attached thereto. See, e.g., Cook et al.,International Publication WO 94/19023 (hereby incorporated by referenceherein).

Other chemical modifications of oligonucleotides (e.g. probes, effectoroligonucleotides) useful in facilitating the delivery of theoligonucleotides into cells include, but are not limited to,cholesterol, transduction peptides (e.g., TAT, penetratin, etc.).

One or more of the sugar-phosphodiester type backbone, 2′OH and purineor pyrimidine base is modified. In one embodiment, the deoxyribosebackbone is replaced by peptide nucleic acid.

Effector oligonucleotides can be designed such that at least one of theone or more sites of the mismatched bases in the effectoroligonucleotide relative to their corresponding genomic target is at anyposition within the oligo, including at its 5′ end, 3′ end, or at apoint within the oligo, for instance positioned at a site that islocated at a site from the 5′ end of the oligo that corresponds to 5%,5-10%, 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%,or 90-95% of the total length of the oligo.

The mismatched bases in the effector oligonucleotides may introduce intothe genome an alteration of the DNA sequence that adds, substitutes ordeletes bases from the corresponding genomic target of the effectoroligonucleotide following recombination of the effector oligonucleotideinto the genome. Addition of bases can be used to 1) introduce frameshift mutations, 2) encode additional amino acids at the N-terminus,C-terminus or within the protein encoded by the target genetic sequencewhere this sequence is a protein-coding sequence, 3) modify the promoterof a gene of interest for instance to increase or decrease or toconditionally increase or decrease its activity, 4) modify agene-regulatory or DNA-binding sequence element, 5) introduce silentmutations or optimize codons in the coding sequence of a gene ofinterest, 6) modify splicing recognition sites within the genome.Alteration of bases can be used to 1) alter amino acids at theN-terminus, C-terminus or within the protein encoded by the targetgenetic sequence where this sequence is a protein-coding sequence, 2)modify the promoter of a gene of interest for instance to increase ordecrease or to conditionally increase or decrease its activity, 3)modify a gene-regulatory or DNA-binding sequence element, 5) introducesilent mutations or optimize codons in the coding sequence of a gene ofinterest, 6) modify splicing recognition sites within the genome.Deletion of bases can be used to 1) introduce frame shift mutations, 2)delete amino acids at the N-terminus, C-terminus or within the proteinencoded by the target genetic sequence where this sequence is aprotein-coding sequence, 3) modify the promoter of a gene of interestfor instance to increase or decrease or to conditionally increase ordecrease its activity, 4) modify a gene-regulatory or DNA-bindingsequence element, 5) introduce silent mutations or optimize codons inthe coding sequence of a gene of interest, or 6) modify splicingrecognition sites within the genome.

Exemplary effector oligonucleotides include, without limitation, aneffector oligonucleotide engineered to generate the Δ32 variant of CCR5,wherein the effector oligonucleotide has a sequence selected from thegroup consisting of SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8,SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ IDNO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ IDNO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ IDNO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ IDNO:34, SEQ ID NO:35, SEQ ID NO:36, and SEQ ID NO:37. In someembodiments, the effector oligonucleotide has a sequence selected fromthe group consisting of SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ IDNO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ IDNO:13, SEQ ID NO:14, SEQ ID NO:15, and SEQ ID NO:16. In someembodiments, the effector oligonucleotide has a sequence selected fromthe group consisting of SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ IDNO:8, SEQ ID NO:9. SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ IDNO:15, and SEQ ID NO:16. SEQ ID NOs:5-16 are provided in FIG. 4A, andSEQ ID NOs:17-37 are provided in Example 3.

Compositions comprising effector oligonucleotides are contemplatedwithin the scope of the invention. In some aspects, the compositionincludes a pharmaceutically acceptable excipient. By “pharmaceuticallyacceptable,” it is meant the carrier, diluent or excipient must becompatible with the other ingredients of the formulation and notdeleterious to the recipient thereof. Pharmaceutically acceptableexcipients are well known in the art and include sterile water, isotonicsolutions such as saline and phosphate buffered saline, and otherexcipients known in the art. See, e.g., Remington: The Science andPractice of Pharmacy (19th edition, 1995. Gennavo, ed.).

D. Target Sequences and Regions

Effector oligonucleotides interact with target sequences and/or targetregions. The intended effect is on the target sequence(s) and/or targetregions. In some embodiments, the target sequence is chromosomal DNA. Inother embodiments, the target sequence is genomic DNA.

The target sequence can be any chromosomal or genomic sequence,including a sequence that comprises a gene or a sequence that encodes anmRNA or protein. Target sequences can comprise any gene, including anyknown allelic variant or form of the gene, single-nucleotidepolymorphism (SNP) form of the gene, the full-length or truncated formof the gene, a mutated form of the gene, or a combination of any ofthese. Target sequences can also correspond to a variant of a gene thatis linked or associated with a disease or infection or susceptibility toa disease or infection, wherein corresponding effector oligonucleotidescan be used to correct, alter or eliminate the sequence associated withthe disease or infection or susceptibility to the disease or infection.Target sequences can also comprise DNA sequence that comprises in parthost genomic sequence and that in addition also comprises in partgenomic sequence derived from a foreign source or agent, includingfollowing an integration event, for instance the viral sequenceintroduced into the genome following integration of virally-encodednucleic acid sequence. Variants or forms of a gene that are associatedor linked with a disease or infection or susceptibility to a disease orinfection can include the wild-type or predominant form of the gene, orthey can vary from the wild-type variant or form of the gene by one ormore bases, wherein the variable one or mom bases have a differentsequence, are additional bases or are bases that are missing or thathave been deleted compared to the wild-type or predominant form of thegene.

Target sequences that encode an RNA or protein can encode any RNA orprotein, including an alternatively spliced form of the RNA or protein,or a known genetic variant of the RNA or protein, including a knowngenetic variant of the RNA or protein associated with or linked with adisease or infection or susceptibility to a disease or infection.

The target sequence can also be a chromosomal or genomic DNA sequencethat comprises a regulatory element, including a regulatory element thatcan regulate or influence the expression level of a gene, for instance apromoter or the binding site of a transcription factor. The targetsequence can be a DNA regulatory element that can influence DNA foldingor structure, including genome organization or accessibility. Additionalexamples of target sequences include sequences that comprise exons,introns, consensus sequences for exon-intron recognition sites,sequences that bind proteins, repetitive DNA elements, sequences thatcomprise telomeres or telomeric repeats, sequences that correspond tocentromeres, or sequences that encode RNAs including coding ornon-coding RNAs, structural RNAs, mRNAs, inhibitory RNAs, antisenseRNAs, RNAs that mediate RNA interference and/or RNAs with catalyticactivity. Depending on the nature or type of a target sequence to beedited, the target sequence can comprise any number of bases rangingfrom 5 to 50000, 5 to 10, 10 to 30, 30 to 40, 40 to 100, 100 to 200, 200to 500, 500 to 1000, 1000 to 2000, 2000 to 5000, 5000 to 10,000, 10,000to 15000, 15000 to 20000, 20000 to 50000 or greater than 50000 bases.

In the case of a target sequence that comprises a variant form orsequence of a gene that is correlated, associated or linked to a diseaseor infection or to susceptibility to a disease or infection, effectoroligonucleotides corresponding to the target sequence can be designed tocorrect the variant or to alter it such that its correlation,association or linkage to the disease or infection or susceptibility tothe disease of infection is altered, reduced, or eliminated.

In the case of a target sequence that comprises a variant form orsequence of a gene that is correlated, associated or linked to aphysiological or biological function that is implicated in health ordisease, effector oligonucleotides corresponding to the target sequencecan be designed to alter the variant such that its associated or linkedphysiological or biological function is altered, enhanced, reduced,activated or eliminated.

In the case of a target sequence that comprises a promoter orgene-regulatory element, effector oligonucleotides corresponding to thetarget sequence may be designed for use and used to alter the sequenceto increase, decrease, alter, enhance, or eliminate the function oractivity of the target sequence.

Effector oligonucleotides may be used to alter any target sequence inthe genome by changing the sequence, or adding to or deleting from thesequence. This alteration can comprise 1-100, 1-200, 1-300, 1-400,1-500, or more bases, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11-15,15-20, 20-25, 26-30, 31, 32, 33-40, 41-50, 50-60, 60-70, 70-100, or morethan 100 bases. In some embodiments, the alteration comprisesconsecutive bases, e.g. addition or deletion of a sequence. In someembodiments, the alteration comprises non-consecutive bases, or morethan one set of consecutive bases, for example deletion of one or morebases from different (non-consecutive) locations in the target sequence,or addition and/or deletion of more than one sequence, where the twoadded and/or deleted sequences are not consecutive, or any combinationsthereof.

Target sequences can be in genes or gene regions that are associatedwith, correlate with or cause diseases, or susceptibility to disease orinfection, and other physiological conditions. The effectoroligonucleotides of the invention can be used to target sequences wheretwo or more bases produce disease or result in susceptibility to adisease. In some instances, the target sequence is a virus receptor,such as an HIV receptor. One non-limiting example is Acquired ImmuneDeficiency Syndrome (AIDS) that is caused by human immunodeficiencyvirus (HIV). The effector oligonucleotide target sequence is an HIVreceptor, such as CCR5. In one example, the mismatch in the effectoroligonucleotide is a deletion. For example, the effector oligonucleotidedoes not match a target sequence to be deleted, but matches thesequences in the target that are before and after the sequence to bedeleted. In one example, a 32 base target sequence is deleted in a Δ32variant of CCR5, wherein the Δ32 variant is compared to the wild-typesequence of CCR5. The effector oligonucleotide comprises a sequence thatcomprises matches to the target sequence before the Δ32 sequence to bedeleted and comprises matches to the target sequence after the Δ32sequence to be deleted, but does not match the Δ32 sequence to bedeleted. Such deletions can be similarly effected in any target sequenceto provide the resulting cell having the desired sequence deleted.

E. Methods of Using Effector Oligonucleotides for Genomic Editing

Effector oligonucleotides can be contacted with cells or introduced intocells. Various parameters can be altered to increase the efficiency ofeffector oligonucleotide-mediated alteration of the genome. Parameters,and any combinations thereof, that can be adjusted to increase theefficiency of the process include any of: alteration of the conditionsused to introduce effector oligonucleotides into cells, including use ofcells at particular points in the cell cycle, stimulation of the cellsused, varying methods used to introduce effector oligonucleotides intocells, including electroporation or chemically-mediated oligonucleotideintroduction, varying the density of cells exposed to effectoroligonucleotides, varying the concentration of effectoroligonucleotides, varying incubation times and temperatures used duringintroduction of the effector oligonucleotides and following this step,as well as adding varying concentrations of one or more reagents thoughtto increase nucleic acid introduction into cells, or the recombinationfrequencies or efficiency of effector oligonucleotide recombination withthe genome of cells, during the steps used to introduce effectoroligonucleotides into cells as well as during prior and subsequentincubation steps. PNA reagents or pseudocomplementary oligonucleotides(such as those taught in U.S. Pat. No. 8,309,356, the disclosure ofwhich is hereby incorporated herein by reference) and other moleculesthat target and bind to or in the vicinity of the sequences targeted byeffector oligonucleotides may also be used to increase the efficiency ofeffector oligonucleotide-mediated alteration of the genome.

Cells enriched or selected for cells at particular points in the cellcycle can be used to increase the efficiency of effectoroligonucleotide-mediated alteration of the genome. The organization ofthe genome of a cell and the accessibility of particular genes in a cellmay vary depending on the conditions used to culture cells or the stageor point of the cells in the cell cycle (including mitosis, G0, G1, G2or S-phase or senescence). The cells used may be cultured underdifferent culture conditions or they may be synchronized in order toenrich or select for cells at a particular stage or point in the cellcycle. For instance nutrient-rich or nutrient-depleted media may be usedto culture cells (for instance the concentration of serum, growthfactors, cytokines, sugars, amino acids, vitamins, growth hormones orany one or more of these or other reagents used in tissue culture may bevaried). Additionally, conditioned media may be used or the cells may becultured in the presence of feeder or irradiated cells. The cells usedmay be synchronized at any stage or point of the cell cycle usingchemical agents, by nutrient-deprivation, or by mechanical harvestingmethods. Mechanical harvesting methods to synchronize cells includetaking advantage of the lower adherence of mitotic cells to the surfaceof growth chamber vessels whereby tapping the vessels causes theirpreferential release into culture, which can then be collected to enrichfor mitotic cells. The cells may be used while they are arrested in astage of the cell cycle or following release from such arrest. One ormore of these methods may be applied to any type of cell, providingcells under different conditions or states.

The stimulation of the cells used can also be adjusted to increase theefficiency of effector oligonucleotide-mediated alteration of thegenome. The cells used may be stimulated or treated with chemicals orreagents at any point during the steps of the method, including prior,during or following introduction of effector oligonucleotides intocells, for instance using electroporation. For example, the cells may beexposed to growth factors, cell signaling factors, hormones, cytokines,nutrients, toxins, mutagens, drugs, sugars, ingredients of tissueculture media, serum, proteins, chemicals, small molecules or a mixtureof one or more of these. Such agents may alter the growth properties orstate of the cells, the permeability of the cells, the organization ofthe genome of the cells, the accessibility of target genes within thecells, the expression of genes including target genes by the cells, theviability of the cells or other properties of the cells.

Methods used to introduce effector oligonucleotides into cells,including electroporation or chemically-mediated effectoroligonucleotide introduction can also be adjusted to increase theefficiency of effector oligonucleotide-mediated alteration of thegenome. Depending on cell type, a great number of methods exist that canbe used to introduce molecules such as oligonucleotides, chemicallymodified oligonucleotides and labeled oligonucleotides into cells.Nucleic acids including effector oligonucleotides may be introduced intothe cells using known means. Techniques for introducing nucleic acidsinto cells are well-known and readily appreciated by the skilled worker.The methods include but are not limited to transfection, viral delivery,protein or peptide mediated insertion, coprecipitation methods, lipidbased delivery reagents (lipofection), cytofection, lipopolyaminedelivery, dendrimer delivery reagents, electroporation or mechanicaldelivery. Examples of transfection reagents are GENEPORTER, GENEPORTER2,LIPOFECTAMINE, LIPOFECTAMINE 2000, FUGENE 6, FUGENE HD, TFX-10, TFX-20,TFX-50, OLIGOFECTAMINE, TRANSFAST, TRANSFECTAM, GENESHUTTLE, TROJENE,GENESILENCER, X-TREMEGENE, PERFECTIN, CYTOFECTIN, SIPORT, UNIFECTOR,SIFECTOR, TRANSIT-LT1, TRANSIT-LT2, TRANSIT-EXPRESS, IFECT, RNAISHUTILE, METAFECTENE, LYOVEC, LIPOTAXI, GENEERASER, GENEJUICE, CYTOPURE,JETSI, JETPEI, MEGAFECTIN, POLYFECT, TRANSMESSANGER, RNAiFECT,SUPERFECT, EFFECTENE, TF-PEI-KIT, CLONFECTIN, and METAFECTINE. Inaddition to the use of chemical reagents used to transfect these andother molecules into cells, electroporation may be used. Also,mechanical means may be used, including for instance passing a mixtureof cells in the presence of the molecules to be introduced through asyringe needle several times or by cell scraping, where a cell scraperis used to scrape cells growing in a culture vessel in the presence ofthe molecule to be introduced. Additionally, pore-forming reagents maybe used to create holes in cell membranes through which the molecules tobe introduced may pass. Other methods include nanoparticles that aredesigned to pierce cells, where these may be complexed with or coated bythe molecule to be delivered. Ballistic methods including gene-guns mayalso be used. Passive diffusion of molecules added to culture media mayalso be used. The method of choice depends on the cell type to be usedand extensive teaching in the art exists such that the appropriatemethod for a given cell type or application may be selected.

Another parameter than can be adjusted to increase the efficiency ofeffector oligonucleotide-mediated alteration of the genome is varyingthe density of cells exposed to effector oligonucleotides. Celldensities ranging from 10,000 cells/ml or fewer to 10,000,000 cells/mlor greater may be used. In some embodiments, the cell density rangesfrom 10,000 cells/ml to 100,000,000 cells/ml, 10,000 cells/ml to50,000,000 cells/ml, 10,000 cells/ml to 10,000,000 cells/ml, 50,000cells/ml to 10,000,000 cells/ml, 100,000 cells/ml to 10,000,000cells/ml, 500,000 cells/ml to 10,000,000 cells/ml or 1,000,000 cells/mlto 10,000,000 cells/ml when exposed to the effector oligonucleotide, Theconcentration of cells used may depend on the method that is used tointroduce reagents such as effector oligonucleotides or fluorogenicprobes into cells.

Another parameter than can be adjusted to increase the efficiency ofeffector oligonucleotide-mediated alteration of the genome is theconcentration of effector oligonucleotides. The concentration ofeffector oligonucleotides to be used can range from nM to mMconcentrations, for example ranging from 1 nM to 1000 mM, 50 nM to 100mM, or 100 nM to 1 mM. In most cases, effector oligonucleotides would beused at μM concentrations, for example 0.1 μM to 1000 μM, 0.1 to 100 μM,0.1 to 50 μM, 0.1 to 10 μM, or 0.1 to 1 μM. The exact concentration ofthe effector oligonucleotide used can be optimized empirically dependingon factors including the cell type and method of delivery used, as wellas for instance the accessibility of the target or the efficiency withwhich a particular effector oligonucleotide is recombined into thegenome at its corresponding target site.

Efficiency of effector oligonucleotide-mediated alteration of the genomecan also be increased by varying incubation times used duringintroduction of the effector oligonucleotide into cells. In methods usedto expose or contact cells to effector oligonucleotides or to introduceeffector oligonucleotides into cells, there are a number of possibleincubation steps the length or duration of any of which can be varied,including but not limited to: 1) depending on the delivery method used,reagents used to mediate or facilitate oligonucleotide delivery may beincubated or pre-incubated with the effector oligonucleotide, 2) thecells may be incubated with effector oligonucleotide, possibly in thepresence of other reagents, and 3) the cells may be incubated with theeffector oligonucleotide following steps such as electroporation,chemically-mediated or mechanical methods used to introduce the effectoroligonucleotides. These incubations may be from 1 to 5 minutes to 1 to 5hours to overnight or longer. For instance, following the step used tointroduce effector oligonucleotides into cells, the cells may beincubated or cultured without removal of the effector oligonucleotidesor the media used during this step. In this case, the cells may beincubated for multiple days in the same medium used during this step, oradditional media may be added at some later point following at least 1to 2 hours to 1 to 5 days or more time in culture.

Efficiency of effector oligonucleotide-mediated alteration of the genomecan also be increased by varying temperatures used during one or more ofthe incubation steps. In some embodiments, for the incubations describedherein, the temperature may vary from ice cold (i.e., 0 to 4° C.) to 65°C., depending on the length of incubation. Generally, incubationtemperatures that are compatible with cell viability range from ice coldto 37° C. or 37-45° C. Higher incubations may also be used, for instanceup to or greater than 45° C. to 70° C., however, depending on the celltype, these incubations must be from 1 to 5 minutes to less than 60minutes in order to preserve cell viability. In most cases, incubationswill range in temperature from ice cold to 37° C.

Another parameter than can be adjusted to increase the efficiency ofeffector oligonucleotide-mediated alteration of the genome is by addingvarying concentrations of one or more reagents thought to increasenucleic acid introduction into cells or to increase recombinationfrequencies or efficiencies in cells. Reagents that may be used includeDMSO or other solvents that are thought to play a role in cellpermeability and oligonucleotide/DNA/genome structure/accessibility orthe association between two or more nucleic acids, including effectoroligonucleotides and target sequences. In the case of DMSO,concentrations ranging from 0.1% to 5% would be most preferred, althoughfor shorter incubations concentrations up to 10% or higher may be used.Other reagents to be used here include polyethelylene glycol, heparinand/or dextrans of varying lengths.

The number of effector oligonucleotide used may be varied to increasethe efficiency of effector oligonucleotide-mediated alteration of thegenome. In one embodiment, two or more effector oligonucleotides todifferent genes may be used. Alternatively, two or more effectoroligonucleotides to the same gene but directed to different portions ofthe same gene may be used, where detection of cells positive forrecombination mediated by each of these different effectoroligonucleotides may be used to enrich for cells having had undergonerecombination at both chromosomal loci of the target gene. Additionally,two or more effector oligonucleotides designed to alter the same pointor sequence on the same gene or target may be used, where for instanceeach of the effector oligonucleotides may be varied in length or bychemical modification. In this case, the effector oligonucleotides wouldall be designed to correspond to one strand of the target sequence, suchthat they do not hybridize to each other.

1. Cells for Use with Effector Oligonucleotides

As shown in exemplary figures, FIG. 5 shows results for two mammaliancell types, K562 and THP cells, treated with the effectoroligonucleotides described in FIG. 4. The steps and methods used tointroduce effector oligonucleotides into cells entail known methods inthe art, and analogous methods exist to introduce effectoroligonucleotides into other cell types. The steps relating to effectoroligonucleotide-mediated recombination subsequent to introduction ofeffector oligonucleotides into the cells are not cell type specific.Consequently, the methods described here can be applied to additionalcell types, including cell types of bacterial and eukaryotic origin,including mammalian, human, animal, plant, yeast, insect, reptilianorigin. Mammalian cell types that may be used include stem cells andadult or differentiated cells of various origins. Stem cell types thatmay be used include embryonic stem cells, induced pluripotent stemcells, adult stem cells, hematopoietic stem cells, adult stem cellsderived from or of various tissues or organs of various types, cordblood stem cells, and cancer stem cells. Additional cell types includemultipotent progenitor cells, lineage-restricted progenitor cells,common myeloid progenitor cells, Granulocyte-macrophage progenitorcells, and megakaryocyte-erythroid progenitor cells. Differentiated celltypes that may be used include immune cells, muscle cells, cardiacmuscle cells, cells of the eye, skin cells, hair cells, epithelialcells, lung cells, kidney cells, interstitial cells, neuronal cells andany other cell type that is found in the body.

Any desired cell type may be used for the cells of the invention. Thecells may be prokaryotic or eukaryotic. The cells may express theprotein of interest (i.e. a protein that is to be altered by treatmentwith the effector oligonucleotide) in their native state or not.Eukaryotic cells that may be used include but are not limited to fungicells such as yeast cells, plant cells and animal cells. Animal cellsthat can be used include but are not limited to mammalian cells andinsect cells, and primary or immortalized cells derived from mesoderm,ectoderm or endoderm layers of eukaryotic organisms. The cells may beendothelial, epidermal, mesenchymal, neural, renal, hepatic,hematopoietic, or immune cells. For example, the cells may be intestinalcrypt or villi cells, clara cells, colon cells, intestinal cells, gobletcells, enterochromafin cells, enteroendocrine cells. Mammalian cellsthat are useful in the method include but are not limited to human,non-human primate, cow, horse, goat, sheep, pig, rodent (including rat,mouse, hamster, guinea pig), marsupial, rabbit, dog and cat. The cellscan be differentiated cells or stem cells, including embryonic stemcells.

Cells of the invention can be primary, transformed, oncogenicallytransformed, virally transformed, immortalized, conditionallytransformed, explants, cells of tissue sections, animals, plants, fungi,protists, archaebacteria and eubacteria, mammals, birds, fish, reptiles,amphibians, and arthropods, avian, chicken, reptile, amphibian, frog,lizard, snake, fish, worms, squid, lobster, sea urchin, sea slug, seasquirt, fly, squid, hydra, arthropods, beetles, chicken, lamprey,ricefish, zebra finch, pufferfish, and Zebrafish.

Additionally, cells such as blood/immune cells, endocrine (thyroid,parathyroid, adrenal), GI (mouth, stomach, intestine), liver, pancreas,gallbladder, respiratory (lung, trachea, pharynx), Cartilage, bone,muscle, skin, hair, urinary (kidney, bladder), reproductive (sperm,ovum, testis, uterus, ovary, penis, vagina), sensory (eye, ear, nose,mouth, tongue, sensory neurons), Blood/immune cells such as_B cell, Tcell (Cytotoxic T cell, Natural Killer T cell, Regulatory T cell, Thelper cell, γδ Tcell, Natural killer cell; granulocytes (basophilgranulocyte, eosinophil granulocyte, neutrophilgranulocyte/hypersegmented neutrophil), monocyte/macrophage, red bloodcell (reticulocyte), mast cell, thrombocyte/Megakaryocyte, dendriticcell; endocrine cells such as: thyroid (thyroid epithelial cell,parafollicular cell), parathyroid (parathyroid chief cell, oxyphilcell), adrenal (chromaffin cell), nervous system cells such as: glialcells (astrocyte, microglia), magnocellular neurosecretory cell,stellate cell, nuclear chain cell, boettcher cell, pituitary,(gonadotrope, corticotrope, thyrotrope, somatotrope, lactotroph),respiratory system cells such as pneumocyte (type I pneumocyte, type IIpneumocyte), clara cell, goblet cell; circulatory system cells such asmyocardiocyte ⋅ pericyte; digestive system cells such as stomach(gastric chief cell, parietal cell), goblet cell, paneth cell, G cells,D cells, ECL cells, I cells, K cells, enteroendocrine cells,enterochromaffin cell, APUD cell, liver (hepatocyte, kupffer cell),pancreas (beta cells, alpha cells), gallbladder,cartilage/bone/muscle/integumentary system cells such as osteoblast,osteocyte, steoclast, tooth cells (cementoblast, ameloblast), cartilagecells: chondroblast, chondrocyte, skin/hair cells: trichocyte,keratinocyte, melanocyte, muscle cells: myocyte, adipocyte, fibroblast,urinary system cells such as podocyte, juxtaglomerular cell,intraglomerular mesangial cell/extraglomerular mesangial cell, kidneyproximal tubule brush border cell, macula densa cell; reproductivesystem cells such as spermatozoon, sertoli cell, leydig cell, ovum,ovarian follicle cell; sensory cells such as organ of corti cells,olfactory epithelium, temperature sensitive sensory neurons, merckelcells, olfactory receptor neuron, pain sensitive neurons, photoreceptorcells, taste bud cells, hair cells of the vestibular apparatus, carotidbody cells are useful to make cells or cell lines of the invention.

Plant cells that are useful include roots, stems and leaves and planttissues include meristematic tissues, parenchyma collenchyma,sclerenchyma, secretory tissues, xylem, phloem, epidermis, periderm(bark).

Cells that are useful for the cells and cell lines of the invention alsoinclude but are not limited to: Chinese hamster ovary (CHO) cells,established neuronal cell lines, pheochromocytomas, neuroblastomasfibroblasts, rhabdomyosarcomas, dorsal root ganglion cells, NS0 cells,CV-1 (ATCC CCL 70), COS-1 (ATCC CRL 1650), COS-7 (ATCC CRL 1651), CHO-K1(ATCC CCL 61), 3T3 (ATCC CCL 92), NIH/3T3 (ATCC CRL 1658), HeLa (ATCCCCL 2), C1271 (ATCC CRL 1616), BS-C-1 (ATCC CCL 26), MRC-5 (ATCC CCL171). L-cells, HEK-293 (ATCC CRL1573) and PC12 (ATCC CRL-1721), HEK293T(ATCC CRL-11268), RBL (ATCC CRL-1378), SH-SYSY (ATCC CRL-2266), MDCK(ATCC CCL-34), SJ-RH30 (ATC CRL-2061), HepG2 (ATC HB-8065), ND7/23(ECACC 92090903), CHO (ECACC 85050302), Vero (ATCC CCL 81), Caco-2 (ATCHTB 37), K562 (ATCC CCL 243), Jurkat (ATCC TIB-152), Per.C6 (Crucell,Leiden, The Netherlands), Huvec (ATCC Human Primary PCS 100-010, MouseCRL 2514, CRL 2515, CRL 2516), HuH-7D12 (ECACC 01042712), 293 (ATCC CRL10852), A549 (ATCC CCL 185), IMR-90 (ATCC CCL 186), MCF-7 (ATC HTB-22),U-2 OS (ATCC HTB-96), T84 (ATC CCL 248), or any established cell line(polarized or nonpolarized) or any cell line available from repositoriessuch as American Type Culture Collection (ATCC, 10801 University Blvd.Manassas, Va. 20110-2209 USA) or European Collection of Cell Cultures(ECACC, Salisbury Wiltshire SP4 OJG England).

Further, cells that are useful in the method of the invention aremammalian cells amenable to growth in serum containing media, serum freemedia, fully defined media without any animal-derived products, andcells that can be converted from one of these conditions to another.

II. Composition of Stem Cells and Methods for Modifying Stem Cells

The invention provides for a composition of stem cells and/or apopulation of stem cells that have been engineered using an effectoroligonucleotide as described herein. In some instances, the stem cellshave been engineered to be refractory to HIV-infection. In one example,this is accomplished by introducing a mutation in the CCR5 gene thatcreates a frameshift mutation. In one embodiment, the CCR5 variant has32 bases deleted from CCR5 to create a CCR5 variant. Specifically, theΔ32 variant of CCR5 is created by a deletion of 32 bases from the gene,resulting in a frameshift mutation. This deletion occurs downstream ofthe start codon at a position such that the preceding 52% of CCR5 plusan additional 31 novel carboxy-terminal amino acids encoded by theframeshift mutation persist. In other embodiments, a CCR5 variant isgenerated such that less than 32 bases is deleted but still results in aframeshift mutation such that additional novel carboxy-terminal aminoacids are encoded by the frameshift mutation.

Stem cells that are used for the invention can be obtained fromdifferent starting sources including, but not limited to, peripheralblood, cell cultures, adult or fetal tissues. The stem cells that areused for the invention for molecular manipulation are cells that haveself-regeneration capacities and the ability to become another type ofcell in its differentiation pathway. The stem cells that are used as astarting source can be totipotent, pluripotent, multipotent, oligopotentand/or unipotent. In some embodiments, the cells are obtained fromperipheral blood. In other embodiments, a treatment is used to mobilizeimmune stem cells into the peripheral blood before the blood iscollected, including, but not limited to, treatment with a cytokine suchas G-CSF. In other embodiments, the cells are obtained from umbilicalcord blood.

In other embodiments, the starting course of stem cells can be inducedpluripotent stem cells (iPS cells of iPSC). As is known to one of skillin the art, iPS cells can be obtained by introduction of a cocktail ofgenes into or with differentiated cells. The cocktail of genes includesbut is not limited to Oct 3/4, Sox2, Nanog, c-Myc and LIN-28.Alternatively, a cocktail of chemicals known to one of skill in the art,structurally related compounds, functionally related compounds or acombination thereof can be used to generate iPS cells. See, for example,Hou P., et al. Science Vol. 341 No. 6146 pp. 651-654; Aug. 9, 2013. Insome embodiments, the piggyback transposable system is used to introducethe cocktail of genes for a traceless production of iPSC where the genescan be removed following their desired activity with no DNA foot-printor DNA residues left behind on the genome of the treated cells. See, forexample, Lacoste, A., et al., Cel Stem Cell. 2009 Sep. 4; 5(3):332-42.doi: 10.1016/j.stem.2009.07.011.

In other embodiments, the cells are obtained from adult or fetaltissues, including an organ, the bone marrow, spleen, thymus or liver.Methods for isolating various types of stem cells from these organs areknown to one of the skill in the art.

Other non-limiting examples of starting cells that can be used include:stem cells which are or are not themselves subject to infection by HIV,stem cells where the genetic locus comprising the CCR5 gene is or is nottranscriptionally active in the stem cells.

In other embodiments, the stem cell types may be anyone or more of thecells in the group consisting of embryonic stem cells,induced-pluripotent stem cells, hematopoietic stem cells, corid bloodstem cells, multipotent progenitor cells, lineage-restricted progenitorcells, common myeloid progenitor cells, Granulocyte-macrophageprogenitor cells and megakaryocyte-erythroid progenitor cells.

In other embodiments, the stem cells that are used express one or moreof the markers selected from the group consisting of: CD34, CD133,CD105, CD45, CD59, Thy1 (CD90), C-kit (CD117) and SLAM family of cellsurface markers. In some embodiments, the SLAM family of cell surfacemarkers is selected from the group consisting of CD48, CD150, and CD244.In other embodiments, the stem cells do not express one or more of themarkers selected from the group consisting of: CD13, CD33, CD71, CD19,and CD61.

In other embodiments, the stem cells comprise an RNA corresponding to anintracellular, non-cell-surface-localized or a cell-surface localizedstem cell marker. Additional non-limiting examples of these stem cellmarkers include transcription factor gene families, signal pathwaygenes, kinase genes. See for example, Kim Y.C., et al. PNAS Vol.106(20):8278-83 (2009), the contents of which are expressly incorporatedfor the markers, gene expression profiles and gene signatures of CD34⁺hematopoietic stem cells. Non-limiting examples of transcription factorgene families include: zf-C2H2, KRAB,Homeobox, HLH, BTB, SCAN, Hormonereceptor, zf-C4, bZIP, ETS, Fork head, PAS, TIG, PHD, GATA, and MYBDNA-binding. Non-limiting examples of signal pathway genes detected inCD34⁺ hematopoietic cells include: calcium signaling pathway, ERB Bsignaling pathway, Hedgehog signaling pathway, JAK-STAT signalingpathway, MAPK signaling pathway, mTOR signaling pathway, Notch signalingpathway, Phosphatidylinositol signaling system, TGF-beta signalingpathway, VEGF signaling pathway, and WNT signaling pathway. Non-limitingexamples of Kinase genes detected in CD34⁺ hematopoietic cells include:AGC, Atypical, Calcium/calmodulin regulated kinases, Casein kinase,CMGC, Receptor guanylate cyclase, STE, Tyrosine kinase-like, andTyrosine kinases. Other markers and expression profiles that can be usedare disclosed in Liu, X., et. al, J. Leukocyte Biology, Vol 82 (4) pp986-1002, October 2007, the contents of which are expressly incorporatedfor the markers, gene expression profiles and gene signatures of CD34⁺hematopoietic stem cells.

The starting source of stem cells includes stem cell where there is atleast 1 copy of wild type CCR5. That is, the stem cells that contain atleast 1 copy of CCR5 that does not have any CCR5 mutations, CCR5deletions (e.g., resulting in a frameshift), and/or CCR5 truncation. Insome cases, the stem cells contain 2 copies of wild type CCR5. In somecases, the stem cells carry one or two copies of a variant of CCR5 thatencodes a protein that may act as a receptor or co-receptor for HIVdocking or entry.

In addition, the starting source of stem cells includes stem cells thathave the potential to become infected with HIV and/or stem cells thatcan differentiate into cells that could become infected with HIV.Non-limiting examples include stem cells that can become immune cells,such as CD4⁺ T cells and CD4⁺ stem cells.

F. Detection, Selection and/or Isolation of Cells with EffectorOligonucleotide-Mediated Editing

Following treatment of cells with effector oligonucleotides designed toalter the genome of the cells, fluorogenic oligonucleotide probes incombination with fluorescence-activated cell sorting can be used toselectively detect and isolate the rare, optimally engineered cells thatcomprise the altered genetic sequence resulting from recombination ofthe mismatch sequence encoded by the effector oligonucleotide into thegenome. In order to increase the signal to noise for detectingoptimally-engineered cells from non-engineered cells using thefluorogenic probes directed to detect the altered sequence, effectoroligonucleotides designed to introduce greater than one mismatch weretested. Effector oligonucleotides that comprise greater than a singlemismatch were found to result in effector oligonucleotide-mediatedalteration of the genome, an unexpected result in view of the teachingin the art that effector oligonucleotides must be essentially identicalto their corresponding target sequences.

In some embodiments, following effector oligonucleotide-mediatedalteration of the genome in cells, optimally-engineered cells may bedetected and isolated using fluorogenic oligonucleotide probes.Exemplary methods and compositions that can be used for detection and/orisolation include US2006/147937, US2010/212040, US2009/106853, andUS2012/015841 (the contents of which are expressly incorporated hereinby reference for the teaching of fluorogenic oligonucleotide probes, andother compositions for detection and/or isolation of engineered cells).In some embodiments wherein the effector oligonucleotide-mediatedrecombination results in the expression of an altered genetic sequencefollowing recombination events, cells having had undergone recombinationmay be positively detected or isolated using fluorogenic probes that aredesigned to detect and report the presence of the resulting expressedsequence. In some embodiments wherein the effectoroligonucleotide-mediated recombination results in the expression of analtered genetic sequence following recombination events, cells havinghad undergone recombination may be negatively detected or isolated usingfluorogenic probes that are designed to detect and report the presenceof the original, non-recombined expressed sequence. In some embodimentswherein the effector oligonucleotide-mediated recombination results inthe expression of an altered genetic sequence following recombinationevents, cells having had undergone recombination may be positivelydetected or isolated using fluorogenic probes that are designed todetect a target within the expressed sequence that is present in boththe recombined and non-recombined sequence, but that is differentiallydetectable or accessible in the cells comprising the recombinedsequence. In some embodiments wherein the effectoroligonucleotide-mediated recombination event results in the expressionof an altered genetic sequence following recombination events, cellshaving had undergone recombination may be negatively detected usingfluorogenic probes that are designed to detect a target within theexpressed sequence that is present in both the recombined andnon-recombined sequence, but that is differentially detectable oraccessible in the cells comprising the non-recombined sequence. In someembodiments wherein the effector oligonucleotide-mediated recombinationevent results in the increased expression of a genetic sequence, cellshaving had undergone recombination may be positively detected usingfluorogenic probes that are designed to detect and report the presenceof the resulting expressed sequence, wherein cells with an increasedsignal for the probe would be identified. In some embodiments whereinthe effector oligonucleotide-mediated recombination event results in thedecreased expression of a genetic sequence, cells having had undergonerecombination may be negatively detected using fluorogenic probes thatare designed to detect and report the presence of the resultingexpressed sequence, wherein cells with a decreased signal for the probewould be identified.

In any of the examples above, cells that are positively or negativelydetected may be isolated, for instance using fluorescence-activated cellsorting. The isolated cells may be isolated individually or in batch orpools. The isolated cells may be expanded in culture clonally, or inpool or batch. The resulting expanded cell culture can be furtherprocessed to remove the culture media from the resulting cells, and thecells can be resuspended in a suitable media for use in cell therapy.

Cells treated with the effector oligonucleotides or the cells that areisolated cells or the cells expanded from the isolated cells may besubjected to downstream analysis, including PCR, genomic PCR, RT-PCR,DNA sequencing, whole-genome sequencing, in situ hybridization,immunofluorescence, western blotting, functional assays, kinetic assays,or any other downstream testing that is known in the art to study andcharacterize cells or sub-cellular fractions and preparations. Cellstreated with the effector oligonucleotides, the isolated cells, or thecells expanded from the isolated cells may be used for cell therapy orto prepare preparations of therapeutic cells or cellular reagents forcell therapy applications. Cells treated with the effectoroligonucleotides, the isolated cells, the cells expanded from theisolated cells or preparations of any of these may be introduced intohumans or animals for testing or therapeutic applications.

One or more of the downstream testing methods may be used to test thecells treated with effector oligonucleotides, the isolated cells or thecells expanded from the isolated cells for the altered or mismatchedsequence encoded by the effector oligonucleotides used to treat thecells. For instance, PCR or DNA or genomic sequencing may be used forthis purpose. In addition, PCR or DNA or genomic analysis includingwhole-genome analysis or sequencing may be used to test for the presenceof any unintended off-target activity or genome modification followingtreatment with effector oligonucleotide. This testing can be applied tocells expanded from individually isolated effectoroligonucleotide-treated cells or effector oligonucleotide-treated cellsisolated as a pool or batch. Cell or cell preparations that areconfirmed to comprise the intended genomic modification introduced bythe effector oligonucleotide and a low frequency of or no otheralteration of the genome could be identified. These cells could be usedin downstream applications, including cell therapy, where they may beexpected to result in a greater likelihood of efficacy and deceasedlikelihood of side-effects or adverse unintended effects due tooff-target or unintended modification of the genome.

FIG. 4A and Example 3 below describe a series of effectoroligonucleotides each of which comprises a mismatch greater than onebase and that were used or can be used to treat cells to introduce agenomic alteration. Multiple oligos were detected as producing theintended genomic alteration in a portion of the cells that were treated.This set of oligos was targeted to introduce an alteration within theCCR5 gene. Oligos may also be designed to introduce genomic alterationsin other genes or DNA sequences, including non-coding sequences such aspromoters, gene-regulatory elements and DNA binding sites. Any gene orDNA sequence may be targeted according to the methods described.

G. CCR5 Deletion and Populations of Stem Cells with Modified CCR5

The modified stem cells of this invention have an altered CCR5 such thatthere is a frameshift mutation. One exemplary embodiment is shown inFIG. 1 of a mutation where 32 bases of the CCR5 are deleted (boldunderlined sequence in FIG. 1). This is also referred to as “delta-32deletion” or “Δ32 deletion”. The 32 bases that are deleted have thesequence of: 5′-GTCAGTATCAATTCGGAAGAATTTCCAGACA (SEQ ID NO:4). Otherframeshift mutations are contemplated within the scope of the invention,for example, those where 29 bases, 26 bases, etc. are removed such thatit results in a frameshift and where novel carboxy-teminal amino acidsare added as exemplified in FIG. 6. In some embodiments, mutations thatresult in one or more stop codon are excluded from specific embodimentsof the invention.

Oligonucleotides can be used to modify the wild-type variant of CCR5into the Δ32 variant of CCR5 in the genome of stem cells. Theseoligonucleotides are optimized for delivery into stem cells.Considerations for optimization include, but are not limited to, (i)oligonucleotides should be non-toxic and compatible with cell viability,(ii) achieve delivery of a concentration of oligonucleotide into cellsthat results in a precise modification of CCR5, (iii) are fully-definedand amenable to quality control and further optimization, and (iv) yieldstem cells where the ability of the isolated stem cells to give rise tocells of the immune system is not perturbed. Exemplary oligonucleotidesthat can be used are described in greater details in the Examplessection.

In some embodiments, stem cells that are used have been manipulated(e.g. by induction) such that they transiently express CCR5 in immunestem cells without loss of the stem cell function. The induction ofexpression of the CCR5 locus in immune stem cells can be helpful whendetection tools, such as molecular beacon probes (e.g., Chromovert®probes) are used to detect cells comprising the Δ32 CCR5.

Utilizing the methodology described herein, one of skill in the art canmodify the CCR5 sequence in stem cells to create a population ofmodified or recombinant stem cells containing CCR5 modifications (e.g.,Δ32 deletion) such that it is rendered refractory to HIV infection.Cells that differentiate from these stem cells are also renderedrefractory to HIV infection. It is understood that reference to apopulation of cells described herein contemplates and includes isolatedpopulations.

In some embodiments, the invention provides for compositions comprisinga substantially pure population of recombinant stem cells, wherein thestem cells comprise a Δ32 deletion in a CCR5 gene. The deletion can bein one allele or both alleles. In one embodiment, the Δ32 deletedsequence is SEQ ID NO:4.

The invention provides for homogeneous or substantially pure populationsof recombinant stem cells that contain the Δ32 CCR5 deletion andcompositions comprising these populations. Accordingly, in someembodiments, the population contains at least about 5% of the stem cellin the population with the Δ32 deletion. In other embodiments, at leastabout 10%, at least about 15%, at least about 20%, at least about 25%,at least about 30%, at least about 35%, at least about 40%, at leastabout 45%, at least about 50%, at least about 55%, at least about 60%,at least about 65%, at least about 70%, at least about 75%, at leastabout 80%, at least about 85%, at least about 90%, at least about 91%,at least about 92%, at least about 93%, at least about 94%, at leastabout 95%, at least about 96%, at least about 97%, at least about 98%,or at least about 99% of the stem cell in the population with the Δ32deletion. In some embodiments, at least about includes an upper limit ofabout 100%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, or 90%, e.g. atleast about 50% is about 50% to 90%, 50% to 91%, 50% to 92%, 50% to 93%,50% to 94%, 50% to 95%, 50% to 96%, 50% to 97%, 50% to 98%, 50% to 99%,or 50% to 100%.

The homogeneous or substantially pure populations of recombinant stemcells that contain the Δ32 CCR5 deletion can be further characterized byits markers (intracellular and extracellular). The stem cells canexpress one or more markers selected from the group consisting of: CD34,CD133, CD105, CD45, CD59, Thy1/CD90, C-kit (CD117) and SLAM family ofcell surface markers or a combination thereof. In some embodiments, theSLAM family of cell surface markers is selected from the groupconsisting of CD48, CD150, and CD244. In other embodiments, the stemcells do not express one or more of the markers selected from the groupconsisting of: CD13, CD33, CD71, CD19, and CD61.

In other embodiments, the stem cells comprise an RNA corresponding to anintracellular, non-cell-surface-localized or a cell-surface localizedstem cell marker. Additional non-limiting examples of these stem cellmarkers include transcription factor gene families, signal pathwaygenes, kinase genes. Non-limiting examples of transcription factor genefamilies include: zf-C2H2, KRAB,Homeobox, HLH, BTB, SCAN, Hormonereceptor, zf-C4, bZIP, ETS, Fork head, PAS, TIG, PHD, GATA, and MYBDNA-binding. Non-limiting examples of signal pathway genes detected inCD34⁺ hematopoietic cells include: calcium signaling pathway, ERB Bsignaling pathway, Hedgehog signaling pathway, JAK-STAT signalingpathway, MAPK signaling pathway, mTOR signaling pathway, Notch signalingpathway, Phosphatidylinositol signaling system, TGF-beta signalingpathway, VEGF signaling pathway, and WNT signaling pathway. Non-limitingexamples of Kinase genes detected in CD34⁺ hematopoietic cells include:AGC, Atypical, Calcium/calmodulin regulated kinases, Casein kinase,CMGC, Receptor guanylate cyclase, STE, Tyrosine kinase-like, andTyrosine kinases. Other markers and expression profiles that can be usedare disclosed in Liu, X., et. al, J. Leukocyte Biology, Vol 82 (4) pp986-1002, October 2007, the contents of which are expressly incorporatedfor the markers, gene expression profiles and gene signatures of CD34⁺hematopoietic stem cells.

Accordingly, in some embodiments, the population contains at least about5% of the stem cell in the population with the one or more markerprofile (or combination of marker) described above. In otherembodiments, at least about 10%, at least about 15%, at least about 20%,at least about 25%, at least about 30%, at least about 35%, at leastabout 40%, at least about 45%, at least about 50%, at least about 55%,at least about 60%, at least about 65%, at least about 70%, at leastabout 75%, at least about 80%, at least about 85%, at least about 90%,at least about 91%, at least about 92%, at least about 93%, at leastabout 94%, at least about 95%, at least about 96%, at least about 97%,at least about 98%, or at least about 99% of the stem cell in thepopulation with the one or more marker (or combination of marker)profile described above. In some embodiments, at least about includes anupper limit of about 100%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%,or 90%, e.g. at least about 50% is about 50% to 90%, 50% to 91%, 50% to92%, 50% to 93%, 50% to 94%, 50% to 95%, 50% to 96%, 50% to 97%, 50% to98%, 50% to 99%, or 50% to 100%.

Various methods can be used to obtain a substantially pure orhomogeneous population of modified stem cells. Detection tools such asthose involving labels or fluorescence probes (e.g., fluorophores,quenchers, molecular beacon probes and the like) can be used to selectfor the stem cells that have the Δ32 modification. One non-limitingexample of a system that can be used in conjunction with the stem cellsis the Chromovert® technology (see, e.g., U.S. Pat. No. 6,692,965). TheChromovert® technology can be used to rapidly isolate and purify thestem cells that have modified CCR5 (e.g, Δ32 CCR5 deletion). Thistechnology can be used with the optimized probes to detect and isolateimmune stem cells that comprise the Δ32 variant of CCR5 are established,where the conditions: (i) provide the highest signal to noise for Δ32versus wild-type CCR5, (ii) are compatible with detection and isolationof viable cells, and (iii) yield immune stem cells where the ability ofthe isolated stem cells to give rise to cells of the immune system isnot perturbed.

One of skill in the art will appreciate that other methods exist toobtain and/or detect substantially pure or homogeneous population ofmodified cells, such as stem cells. For example, fluorescence-activatedcell sorting (FACS) can be used to select for specific stem cells usingpositive staining or alternatively, to exclude by negative staining(e.g., to discard population of stem cells which are negative for CCR5).

The stem cells of the invention can give rise to all cells of the immunesystem or they can give rise to myeloid (monocytes, macrophages,neutrophils, basophils, eosinophils, erythrocytes, megakaryocytes,platelets, dendritic cells) or lymphoid lineages (T-cells, B-cells,NK-cells). In some embodiments, the stem cells can be myeloid-biased,lymphoid-biased or unbiased with respect to the differentiated celltypes that may be derived from the stem cells.

In some embodiments, the stem cells are modified and allowed todifferentiate into the immune cell types that are normally subject toinfection by HIV. With the modifications described herein, these stemcells and differentiated cells derived from the stem cells may be usedaccording to the invention to produce corresponding cell types or immunecell types that are resistant to infection by HIV. In one embodiment,the differentiated cell type is an immune T-cell. In another embodiment,the differentiated cell type is a cell type wherein the genetic locuscomprising the CCR5 gene is or is not transcriptionally active. In someembodiments, the resulting treated differentiated cells may betrans-differentiated or dedifferentiated into other cell types includinginto stem cell types for subsequent testing or therapeutic use. In oneembodiment, the treated cells of the method may be dedifferentiated intohematopoietic stem cells.

H. Methods of Treating Individuals with Recombinant Cells Produced byEffector Oligonucleotide-Mediated Alteration

The population of modified cells (i.e. recombinant cells) as describedherein can be expanded by standard cell culture for cells that are knownto one of the skill in art. The population of modified cells can bemixed with a pharmaceutically acceptable carrier to create apharmaceutical composition suitable for cell therapy. The pharmaceuticalcomposition comprising modified cells, such as stem cells, as describedherein, include at least one pharmaceutically acceptable adjuvant and/orexcipient, as are well known to those skilled in the art. The populationof modified cells can be tested in non-human animal models of thedisease to be treated to determine the effective amount or range ofeffective amount of modified cells that can be used. Non-human animalmodels can also be used to evaluate the retention of cell function bythe population of modified cells.

As a non-limiting example, a population of modified stem cells asdescribed herein can be expanded by standard cell culture for stem cellsthat are known to one of the skill in art. The population of modifiedstem cells can be mixed with a pharmaceutically acceptable carrier tocreate a pharmaceutical composition suitable for cell therapy. Thepopulation of modified stem cells can be tested in non-human animalmodels of HIV to determine the effective amount or range of effectiveamount of modified stem cells that can be used. Non-human animal modelsof HIV can also be used to evaluate the retention of stem cell functionby the population of modified stem cells.

As noted above, the stem cells can differentiate into one or more celltypes, in particular, when introduced in vivo and exposed to thebiological milieu of growth factors, cytokines and other biologicalfactors that would induce a stem cell to differentiate. Without beingbound by theory, the population of modified stem cells can self-renew togenerate an endless supply of modified stem cells as well asdifferentiate into cells that are refractory to HIV infection. Examplesof such differentiated cells include, but are not limited to, immunecell types that are normally otherwise subject to infection by HIV. Inone embodiment, the differentiated cell type is an immune T-cell. Inanother embodiment, the differentiated cell type is a cell type whereinthe genetic locus comprising the CCR5 gene is or is nottranscriptionally active.

In addition, the population of modified cells (e.g. stem cells) can betested to determine the genomic integrity and to assess if there are anyunintended (and/or undesired) genomic modifications. One of the skill inthe can use any molecular biology techniques to detect and measure thefrequency of aberrant genome modification. This assessment can beimportant to ascertain the safety of cell therapy using variouspopulations of modified cells, such as modified stem cells.

The population of modified stem cells can be tested to determine ifcertain cohorts of individuals provide cells that comprise idealcharacteristics for successful cell therapy. Non-limiting examples ofcharacteristics that can be assessed include: markers (intracellular andextracellular), longevity in cell culture, renewal potential, expansionrate, gene expression profiles, proteomics and functionality (e.g.,ability to differentiate into certain types of cells).

Analysis of the characteristics of populations of stem cells fromcertain cohorts may be beneficial when designing treatment plans,including using the cell therapy as primary treatment as well as usingit to aid in the treatment of HIV infection. Certain patentsub-populations can be identified as being particularly receptive totreatment with a population of modified stem cells.

I. Methods of Treating Individuals with Effector Oligonucleotides

In human or animal cell therapy applications, preferred geneticsequences that may be selected to serve as targets for effectoroligonucleotide-mediated alteration of the genome include sequencesimplicated in disease or infection, or susceptibility to disease orinfection. In particular, genetic sequences or variations of two or morebases that produce disease or result in susceptibility to a disease orinfection would be preferred target sequences for modification byeffector oligonucleotide-mediated recombination. Examples of suchsequences include the CCR5 gene or locus, where alteration of thewild-type sequence in cells of the immune system from individualscomprising wild-type CCR5 can be used as part of a cell therapy forHIV/AIDS. The alteration of CCR5 can be designed to mimic, replicate orapproximate the Δ32 sequence variant of CCR5. Numerous diseases orinfections or susceptibilities to disease or infection exist that resultfrom one or more known DNA variations or mutations. These sequencesrepresent preferred targets for alteration using effectoroligonucleotide-mediated recombination.

The effector oligonucleotides can also be used to treat a subject inneed thereof, i.e. rather than treating a subject by cell therapymethods using a recombinant cell that has been altered by treatment withan effector oligonucleotide, the effector oligonucleotide can beadministered to the subject to mediate the alteration of target cellswithin the subject. The invention provides for the use of an effectoroligonucleotide to correct, alter or eliminate the sequence associatedwith a disease or infection or susceptibility to the disease orinfection, wherein the effector oligonucleotide is targeted to a variantof a gene or portion thereof, that is linked or associated with adisease or infection or susceptibility to a disease or infection. Assuch, the invention provides for a method of treating a disease orinfection, or susceptibility to the disease or infection, in a subjectcomprising administering to the subject in need thereof an effectiveamount of an effector oligonucleotide. In some embodiments of the methodof treating a disease or infection in a subject, the effectoroligonucleotide is targeted to a variant of a gene or portion thereof,that is linked or associated with the disease or infection.

J. Pharmaceutically Acceptable Compositions

The effector oligonucleotides for use in the methods of treatment asdescribed herein, e.g. treatment by administering the one or moreeffector oligonucleotides as disclosed herein, can be administered inthe form of pharmaceutical compositions. These effector oligonucleotidesand pharmaceutical compositions thereof can be administered by a varietyof routes including oral, rectal, cerebrospinal, transdermal,subcutaneous, topical, transmucosal, nasopharangeal, pulmonary,intravenous, intraperitonial, intramuscular, and intranasal. In someembodiments, the effector oligonucleotides are administeredsystemically, wherein the administration is intravenous orintraperitoneal administration.

Thus, provided herein are pharmaceutical compositions which contain, asthe active ingredient, one or more of the effector oligonucleotides asdescribed herein associated with one or more pharmaceutically acceptableexcipients or carriers. In making the compositions of this invention,the active ingredient is usually mixed with an excipient or carrier,diluted by an excipient or carrier or enclosed within such an excipientor carrier which can be in the form of a capsule, sachet, paper or othercontainer. When the excipient or carrier serves as a diluent, it can bea solid, semi-solid, or liquid material, which acts as a vehicle,carrier or medium for the active ingredient. Thus, the compositions canbe in the form of tablets, pills, powders, lozenges, sachets, cachets,elixirs, suspensions, emulsions, solutions, syrups, aerosols (as a solidor in a liquid medium), ointments containing, for example, up to 10% byweight of the active compound, soft and hard gelatin capsules,suppositories, sterile injectable solutions, and sterile packagedpowders.

Some examples of suitable excipients or carriers include lactose,dextrose, sucrose, sorbitol, mannitol, starches, gum acacia, calciumphosphate, alginates, tragacanth, gelatin, calcium silicate,microcrystalline cellulose, polyvinylpyrrolidone, cellulose, sterilewater, syrup, and methyl cellulose. The formulations can additionallyinclude: lubricating agents such as talc, magnesium stearate, andmineral oil; wetting agents; emulsifying and suspending agents;preserving agents such as methyl- and propylhydroxy-benzoates;sweetening agents; and flavoring agents. The compositions of theinvention can be formulated so as to provide quick, sustained or delayedrelease of the active ingredient after administration to the patient byemploying procedures known in the art.

In another aspect, one or more effector oligonucleotides as describedherein are encapsulated within a microcarrier for deliver to anindividual. In some embodiments the microcarrier encapsulates more thanone effector oligonucleotide species. In some embodiments, the one ormore effector oligonucleotide species encapsulated within themicrocarrier comprises a nucleic acid sequence selected from the groupconsisting of SEQ ID NOs:5-37. Methods of encapsulating oligonucleotidesin microcarriers are well known in the art, and described, for example,in International application WO98/55495. Colloidal dispersion systems,such as microspheres, beads, macromolecular complexes, nanocapsules andlipid-based system, such as oil-in-water emulsions, micelles, mixedmicelles and liposomes can provide effective encapsulation ofoligonucleotides within microcarrier compositions. The encapsulationcomposition may further comprise any of a wide variety of components.These include, but are not limited to, alum, lipids, phospholipids,lipid membrane structures (LMS), polyethylene glycol (PEG) and otherpolymers, such as polypeptides, glycopeptides, and polysaccharides.

In some embodiments, the compositions can be formulated in a unit dosageform, each dosage containing from about 1 mg to about 1000 mg or more,such as any of about 1 mg to about 900 mg, about 1 mg to about 800 mg,about 1 mg to about 700 mg, about 1 mg to about 600 mg, about 1 mg toabout 500 mg, about 5 mg to about 1000 mg, about 5 mg to about 900 mg,about 5 mg to about 800 mg, about 5 mg to about 700 mg, about 5 mg toabout 600 mg, or about 5 mg to about 500 mg, inclusive, including anyrange in between these values, of the active ingredient, i.e. one ormore effector oligonucleotides. In some embodiments, the compositionscan be formulated in a unit dosage form, each dosage containing fromabout 1 μg to about 1000 μg or more, such as any of about 1 μg to about900 μg, about 1 μg to about 800 μg, about 1 μg to about 700 μg, about 1μg to about 600 μg, about 1 μg to about 500 μg, about 5 μg to about 1000μg, about 5 μg to about 900 μg, about 5 μg to about 800 μg, about 5 μgto about 700 μg, about 5 μg to about 600 μg, or about 5 μg to about 500μg, inclusive, including any range in between these values, of theactive ingredient, i.e. one or more effector oligonucleotides. The term“unit dosage forms” refers to physically discrete units suitable asunitary dosages for subjects, each unit containing a predeterminedquantity of active material calculated to produce the desiredtherapeutic effect, in association with a suitable pharmaceuticalexcipient or carrier.

The therapies disclosed herein are effective over a wide dosage rangeand are generally administered in a therapeutically effective amount. Itwill be understood, however, that the amount of the effectoroligonucleotide actually administered will be determined by a physician,in the light of the relevant circumstances, including the condition tobe treated, the chosen route of administration, the actual compoundadministered, the age, weight, and response of the individual subject,the severity of the subject's symptoms, and the like.

For preparing solid compositions such as tablets, the effectoroligonucleotide is mixed with a pharmaceutical excipient or carrier toform a solid preformulation composition containing a homogeneous mixtureof an effector oligonucleotide of the present invention. When referringto these preformulation compositions as homogeneous, it is meant thatthe active ingredient is dispersed evenly throughout the composition sothat the composition can be readily subdivided into equally effectiveunit dosage forms such as tablets, pills and capsules.

The tablets or pills of the present invention can be coated or otherwisecompounded to provide a dosage form affording the advantage of prolongedaction and to protect the effector oligonucleotide from acid hydrolysisin the stomach. For example, the tablet or pill can comprise an innerdosage and an outer dosage component, the latter being in the form of anenvelope over the former. The two components can be separated by anenteric layer which serves to resist disintegration in the stomach andpermit the inner component to pass intact into the duodenum or to bedelayed in release. A variety of materials can be used for such entericlayers or coatings, such materials including a number of polymeric acidsand mixtures of polymeric acids with such materials as shellac, cetylalcohol, and cellulose acetate.

The liquid forms in which the compositions of the present invention canbe incorporated for administration orally or by injection includeaqueous solutions, suitably flavored syrups, aqueous or oil suspensions,and flavored emulsions with edible oils such as corn oil, cottonseedoil, sesame oil, coconut oil, or peanut oil, as well as elixirs andsimilar pharmaceutical vehicles.

Parenteral routes of administration include but are not limited todirect injection into a central venous line, intravenous, intramuscular,intraperitoneal, intradermal, or subcutaneous injection. Formulationssuitable for parenteral administration (e.g., an effectoroligonucleotide as described herein in a microcarrier formulation) aregenerally formulated in USP water or water for injection and may furthercomprise pH buffers, salts bulking agents, preservatives, and otherpharmaceutically acceptable excipients. Effector oligonucleotides asdescribed herein, for example as microcarrier complexes or encapsulates,for parenteral injection may be formulated in pharmaceuticallyacceptable sterile isotonic solutions such as saline and phosphatebuffered saline for injection.

Compositions for inhalation or insufflation include solutions andsuspensions in pharmaceutically acceptable, aqueous or organic solvents,or mixtures thereof, and powders. The liquid or solid compositions cancontain suitable pharmaceutically acceptable excipients as describedherein. The compositions can be administered by the oral or nasalrespiratory route for local or systemic effect. Compositions inpharmaceutically acceptable solvents can be nebulized by use of inertgases. Nebulized solutions can be inhaled directly from the nebulizingdevice or the nebulizing device can be attached to a face mask tent, orintermittent positive pressure breathing machine. Solution, suspension,or powder compositions can also be administered, orally or nasally, fromdevices which deliver the formulation in an appropriate manner.

K. Articles of Manufacture and Kits

The invention also provides kits and articles of manufactures for use inthe instant methods. Kits of the invention include one or morecontainers comprising one or more effector oligonucleotides as describedherein and instructions for use in accordance with any of the methods ofthe invention described herein.

In some embodiments, these instructions comprise a description ofadministration of the effector oligonucleotides for treating HIVinfection, AIDS, or any disease where a genetic variation of two or morebases causes or is associated with the disease or susceptibility to thedisease or infection. The kit may further comprise a description ofselecting cells or an individual suitable for treatment based onidentifying whether the cells or that individual has the disease and thestage of the disease or the susceptibility to the infection.

The instructions relating to the use of the effector oligonucleotidescan generally include information as to dosage, dosing schedule, androute of administration for the intended treatment. The containers maybe unit doses, bulk packages (e.g., multi-dose packages) or sub-unitdoses. Instructions supplied in the kits of the invention are typicallywritten instructions on a label or package insert (e.g., a paper sheetincluded in the kit), but machine-readable instructions (e.g.,instructions carried on a magnetic or optical storage disk) are alsoacceptable.

The label or package insert can indicate that the composition is usedfor treating susceptibility to HIV infection, HIV infection, AIDS, orany disease where a genetic variation of two or more bases causes or isassociated with the disease or any other uses described herein.Instructions may be provided for practicing any of the methods describedherein.

The kits of this invention are in suitable packaging. Suitable packagingincludes, but is not limited to vials, bottles, jars, flexible packaging(e.g., sealed Mylar or plastic bags), and the like. Also contemplatedare packages for use in combination with a specific device, such assyringe, pre-filled syrine, electroporation cuvetter, an inhaler, nasaladministration device (e.g., an atomizer) or an infusion device such asa minipump. A kit may have a sterile access port (for example thecontainer may be an intravenous solution bag or a vial having a stopperpierceable by a hypodermic injection needle). The container may alsohave a sterile access port (for example the container may be anintravenous solution bag or a vial having a stopper pierceable by ahypodermic injection needle). At least one active agent in thecomposition is an effector oligonucleotide as described herein. Thecontainer may further comprise a second pharmaceutically active agent.The kit may comprise components suitable for treating cells to preparecells with therapeutic potential, including components to isolate thecells to be treated from an individual to be treated or a donorindividual, components to contact the cells with effectoroligonucleotide, and components for use in the detection and isolationof cells treated with the effector oligonucleotide to comprise sequenceencoded by the effector oligonucleotide. The components of the kit mayinclude buffers and reagents for electroporation, fluorescence-activatedcell sorting and for the culture and expansion of cells.

Kits may optionally provide additional components such as buffers andinterpretive information. Normally, the kit comprises a container and alabel or package insert(s) on or associated with the container.

Throughout this specification, various patents, patent applications andother types of publications (e.g., journal articles) are referenced. Thedisclosure of all patents, patent applications, and publications citedherein are hereby incorporated by reference in their entirety for allpurposes.

The invention can be more fully further understood by reference to thefollowing examples, which are provided by way of illustration and arenot meant to be limiting of the scope of the invention. All citationsthroughout the disclosure are hereby expressly incorporated byreference.

EXAMPLES Example 1: Electroporation of Effector Oligonucleotides intoTHP or K562 Cells

THP or K562 cells were cultured to a maximum density of 2×10⁶ per mL inRPMI culture medium (Gibco CAT 61870-036)) supplemented with 10% FBS(SAFC Biosciences CAT 12103C-500ML). The cells were rinsed withserum-free RPMI and resuspended at either 10×10⁶ or 5×10⁶ cells/mL.Effector oligonucleotides tested were numbers 1-8, 19 and 20 of FIG. 4A.For each reaction, 10 μL of a 3.2 μM stock solution of the effectoroligonucleotide tested and 7 μL of a stock solution of 100 μM PNAreagent was added to 100 μL of the K562 and THP cells at eitherconcentration was added to one well of 96-well electroporation plate(Biorad CAT 1652681). The PNA used here was the previously described PNA“tcPNA-679” (N-Lys-Lys-Lys-JTJTTJTTJT-OOO-TCTTCTTCTCATTTC-Lys-Lys-Lys-C)(SEQ ID NO:58). The samples were incubated on ice for 30-60 minutes,pulsed one time at 350 volts for 12 MS using the GenePulser MXCell(Biorad) and then incubated again on ice for 15-30 minutes before theywere transferred into wells of a 12-well tissue culture dish using 500μL of culture medium and incubated in a tissue culture incubator at 37°C., 5% CO₂. Genomic DNA was prepared from the samples after 2-3 days(Molecular Cloning Protocol: A Laboratory Manual, Volume 1, Sambrook andRussel, pp. 6.28-6.33) for analysis by PCR.

Example 2: Genomic PCR on Recombinant THP or K562 Cells

PCR was used to amplify a portion of CCR5 genomic DNA purified fromtreated cells of Example 1. The forward and reverse primer used wereATCACTTGGGTGGTGGCTGTGTTTGCGTCTC (SEQ ID NO:59) for the forward primerand AGTAGCAGATGACCATGACAAGCAGCGGCAG (SEQ ID NO:60) for the reverseprimer. The PCR conditions included a first step of 95° C. for 5minutes, and 45 to 50 cycles of the following three steps: 1) 95° C. for30 seconds, 2) 65° C. for 30 seconds, and 3) 72° C. for 20 seconds. 20μl PCR reactions were set up for each sample and overlaid with a drop ofmineral oil. Taq DNA polymerase was used with components atconcentrations according to manufacturer's instructions (Roche, CAT4738225001). A PCR product with a size of approximately 193 bases wasexpected for genomic sequence corresponding to wild-type CCR5 and a PCRproduct with a size of approximately 161 bases was expected for genomicsequence corresponding to the Δ32 variant of CCR5. FIG. 5 shows theresults for K562 cells (top set of lanes) or THP cells (bottom set oflanes), with control wild type (lane 1) or deleted CCR5 (lane 2) withmolecular weight marker (lane 3) shown separately on the bottom centergel. The lanes are labelled across the top with the effectoroligonucleotide number (see FIG. 4A) or control (C, no effectoroligonucleotide added) or marker (M). Lanes 1-10 for both the top andbottom show effector oligonucleotides 1-8, 19 and 20, respectively, withlane 11 as no effector control, and lane 12 as molecular weight marker.Lanes 13-22 for both the top and bottom show effector oligonucleotides1-8, 19 and 20, respectively, with lane 1 as no effector control. Thetop samples (K562 cells) are at a density of ˜4.3×10⁶ cells/mL in lanes1-10 and ˜8.6×10′ cells/mL in lanes 13-22, while the bottom samples (THPcells) are at a density of ˜4.3×10⁶ cells/mL in lanes 13-22 and ˜8.6×10⁶cells/mL in lanes 1-10. The doublet band near the 200 MW marker showthat both the wild type CCR5 and the Δ32 deleted CCR5 are present in thesamples treated with effector oligonucleotides.

Example 3: Effector Oligonucleotides and Methods to Create Δ32 CCR5 inImmune Stem Cells

This example describes various optimized oligonucleotides that are usedto modify the wild-type variant of CCR5 into the Δ32 variant of CCR5 inthe genome of immune stem cells. Exemplary oligonucleotides are shown inFIG. 4A.

Conditions that optimize the delivery of the oligonucleotides intoimmune stem cells are established. The conditions are thatoligonucleotides: (i) are non-toxic and compatible with cell viability,(ii) achieve delivery of a concentration of oligonucleotide into cellsthat results in a precise modification of CCR5, (iii) are fully-definedand amenable to quality control and further optimization, and (iv) yieldimmune stem cells where the ability of the isolated stem cells to giverise to cells of the immune system is not perturbed.

In addition to immune stem cells, immortalized immune cell lines(including, but not limited to THP and K562 cells) are used as a proxyfor immune stem cells. Identification of conditions that apply acrossall these cell types indicates broad applicability of the methods.Genomic PCR of DNA isolated from cells treated using testoligonucleotide and electroporation conditions are used to evaluatetheir effectiveness (See Examples 1 and 2).

The following effector oligonucleotides are tested in addition to theabove:

Key to modifications, oligonucleotides are 5′ to  3′.:BNA (bridged nucleic acids) - indicated with base  underlined6-Thio-2′-deoxyguanosine - indicated by lower  case g8-Amino-2′-deoxyadenosine - indicated by lower   case a(SEQ ID NO: 17) 1) GAAGGTCTTCATTACACCTGCAGCTCTCATTTTCCATACATTAAAGATAGTCATCTTGGGGCTGGTCCTGCCGCTGCTTG (SEQ ID NO: 18) 2)GAAGGTCTTCATTACACCTGCAGCTCTCATTTTCCATACATTAAAGATAGTCATCTTGGGGCTGGTCCTGCCGCTGCTTG (SEQ ID NO: 19) 3)GAAGGTCTTCATTACACCTGCAGCTCTCATTTTCCATACATTAAAGATAGTCATCTTGGGGCTGGTCCTGCCGCTGCTTG (SEQ ID NO: 20) 4)GAAGGTCTTCATTACACCTGCAGCTCTCATTTTCCATACATTAAAGATAGTCATCTTGGGGCTGGTCCTGCCGCTGCTTG (SEQ ID NO: 21) 5)gAAggTCTTCATTACACCTGCAGCTCTCATTTTCCATACATTAAAGATAGTCATCTTGGGGCTGGTCCTGCCGCTGCTTG (SEQ ID NO: 22) 6)gaaggTCTTCATTACACCTGCAGCTCTCATTTTCCATACATTAAAGATAGTCATCTTGGGGCTGGTCCTGCCGCTGCTTG (SEQ ID NO: 23) 7)gAAggTCTTCATTACACCTGCAGCTCTCATTTTCCATACATTAAAGATAGTCATCTTGGGGCTGGTCCTGCCgCTgCTTG (SEQ ID NO: 24) 8)gaaggTCTTCATTACACCTGCAGCTCTCATTTTCCATACATTAAAGATAGTCATCTTGGGGCTGGTCCTGCCGCTGCTTG (SEQ ID NO: 25) 9)GAAGGTCTTCATTACACCTGCAGCTCTCATTTTCCATACATTAAAGATAGTCATCTTGGGGCTGGTCCTGCCGCTGCTTGTCATGGTCATCTGC TACTCG (SEQ ID NO: 26)10) GAAGGTCTTCATTACACCTGCAGCTCTCATTTTCCATACATTAAAGATAGTCATCTTGGGGCTGGTCCTGCCGCTGCTTGTCATGGTCATCTGC TACTCG (SEQ ID NO: 27)11) GAAGGTCTTCATTACACCTGCAGCTCTCATTTTCCATACATTAAAGATAGTCATCTTGGGGCTGGTCCTGCCGCTGCTTGTCATGGTCATCTGC TACTCG (SEQ ID NO: 28)12) GAAGGTCTTCATTACACCTGCAGCTCTCATTTTCCATACATTAAAGATAGTCATCTTGGGGCTGGTCCTGCCGCTGCTTGTCATGGTCATCTGC TACTCG (SEQ ID NO: 29)13) GAAGGTCTTCATTACACCTGCAGCTCTCATTTTCCATACATTAAAGATAGTCATCTTGGGGCTGGTCCTGCCGCTGCTTGTCATGGTCATCTGC TACTCG (SEQ ID NO: 30)14) GAAGGTCTTCATTACACCTGCAGCTCTCATTTTCCATACATTAAAGATAGTCATCTTGGGGCTGGTCCTGCCGCTGCTTGTCATGGTCATCTGC TACTCG (SEQ ID NO: 31)15) GAAGgTCTTCaTTaCACCTGCAGCTCTCATTTTCCATACATTAAAGATAGTCATCTTGGGGCTGGTCCTGCCGCTGCTTGTCATggTCaTCTgC TaCTCG (SEQ ID NO: 32)16) gaaggTCTTCaTTaCACCTGCAGCTCTCATTTTCCATACATTAAAGATAGTCATCTTGGGGCTGGTCCTGCCGCTGCTTGTCATggTCaTCTgC TaCTCg (SEQ ID NO: 33)17) gaaggTCTTCATTACACCTGCAGCTCTCATTTTCCATACATTAAAGATAGTCATCTTGGGGCTGGTCCTGCCGCTGCTTGTCATggTCaTCTGC TACTCg (SEQ ID NO: 34)18) GAAGGTCTTCATTACACCTGCAGCTCTCATTTTCCATACATTAAAGATAGTCATCTTGGGGCTGGTCCTGCCGCTgCTTgTCaTggTCaTCTGC TACTCg (SEQ ID NO: 35)19) GAAGGTCTTCATTACACCTGCAGCTCTCATTTTCCATACATTAAAGATAGTCATCTTGGGGCTGGTCCTGCCGCTgCTTgTCaTggTCaTCTGC TACTCg (SEQ ID NO: 36)20) GAAGGTCTTCATTACACCTGCAGCTCTCATTTTCCATACATTAAAGATAGTCATCTTGGGGCTGGTCCTGCCGCTGCTTGTCATGGTCATCTGC TACTCG (SEQ ID NO: 37)21) GAAGGTCTTCATTACACCTGCAGCTCTCATTTTCCATACATTAAAGATAGTCATCTTGGGGCTGGTCCTGCCGCTGCTTGTCATGGTCATCTGC TACTCG

Example 4—Detecting and Isolating Immune Stem Cells Treated to CompriseΔ32 CCR5

This example describes using optimized molecular beacon probes(including, but not limited to Chromovert® probes) to detect and isolateimmune stem cells comprising Δ32 CCR5 created as a result of Examples 1and 2, or similarly treated with effector oligonucleotides of Example 3.This example uses the Chromovert® technology as a specific embodiment,but those skilled in the art will recognize, or be able to ascertainusing no more than routine experimentation, other equivalents to thespecific embodiment and methods discussed herein.

Chromovert® conditions for using the optimized probes to detect andisolate immune stem cells that comprise the Δ32 variant of CCR5 areestablished, where the conditions: (i) provide the highest signal tonoise for Δ32 versus wild-type CCR5, (ii) are compatible with detectionand isolation of viable cells, and (iii) yield immune stem cells wherethe ability of the isolated stem cells to give rise to cells of theimmune system is not perturbed.

In addition to immune stem cells, immortalized immune cell lines(including, but not limited to THP and K562 cells) are used as a proxyfor immune stem cells. Optimal Chromovert® probes and conditions aredetermined by applying test probes and methods to a mixture of cellscomprising either Δ32 or wild-type variants of CCR5, isolating positivecells, and using genomic and RT-PCR methods to assess for any enrichmentof cells expressing the Δ32 variant of CCR5.

The Chromovert® probes are developed by iterative rounds of testingstarting with design and testing of a first set of probes. The probesare designed to correspond to the sequence and be specific for the Δ32variant of CCR5. Specifically, the loop portion of the probe is designedto hybridize to the branch-point sequence within the sequence of the Δ32variant of CCR5 that juxtaposes the bases upstream and downstream of the32-base deletion. In addition, probes are designed to detect portions ofCCR5 sequence that are predicted to be accessible for probe-binding onlyin the context of the sequence of Δ32 variant of CCR5 but not in thecontext of the sequence of wild-type CCR5. Such probes are synthesizedand tested by using cells treated to express either the Δ32 or wild-typevariant of CCR5 and those probes that produce the highest signal tonoise ratios are identified. It is known that CCR5 has two alternative5′ un-translated regions. Probes that can detect the Δ32 variant of CCR5comprising either 5′ un-translated region are identified and prioritizedfor further optimization.

The following parameters are used for probe selection: (i) length ofloop and/or stem and (ii) incorporation and extent of one or morechemically-modified bases in the loop and/or stem. Other parameters andconditions used for developing and optimizing molecular beacon probesare optionally pursued for developing Chromovert® probes that can detectthe Δ32 sequence at the DNA level.

Chromovert® conditions are developed and optimized for immune stemcells, including immortalized immune cell lines. Electroporation methodsare optimized for this application and specific cell types. Thefollowing parameters are used for optimizing the electroporationmethods: (i) voltage and duration of electroporation, (ii) number ofpulses, (iii) electroporation buffer, (iv) temperature, (v)concentration of cells, (vi) concentration of oligonucleotide, and (vii)addition of reagents that may improve the efficiency of the overallprocess, including DMSO and other solvents. As those skilled in the artwill recognize, other parameters and conditions may also be used ifnecessary.

The following exemplary probes will be used and tested for furtheroptimization. The length will vary, as will chemical composition, forinstance by incorporation of one or more of the following non-limitinglists of modifications at various positions within the oligo (otherchemical modifications may be used as discussed herein below or known inthe art).

Key to modifications, oligonucleotides are 5′ to 3′:

BNA (bridged nucleic acids)—indicated with base underlined

7-Deaza-8-aza-2′-deoxyadenosine—indicated by lower case a (in probesB15-B18)

5-Propynyl-2′-deoxyuridine—indicated by lower case t (in probes inB15-B19)

2′-Deoxyisoguanosine—indicated by lower case g (in probes B15-B17)

Probe with no modifications: 5′-GCGAGGACTATCTTTAATGTATGGAAAATCTCGC (SEQID NO:38), where the bases in bold target the Δ32-CCR5 recombinantsequence (the bases at termini, i.e. not bold, comprise the stem):

-   -   the bases in the stem may be substituted with other sequences    -   the targeting sequence may correspond to another sequence        specific to Δ32, for instance it may be longer, shorter, or to a        different portion of the region spanning the 32 base deletion,        or it may be to a sequence within the sequence of CCR5 that also        exists in the Δ32 variant of CCR5 but which is differentially        accessible in the Δ32 vs wild-type sequence. Additionally, the        sequences provided are designed to detect the mRNA expressed        from CCR5. For probes to the DNA, probes directed to either        strand of CCR5 may be used.

(SEQ ID NO: 39) B1) GCGAGGACTATCTTTAATGTATGGAAAATCTCGC (SEQ ID NO: 40)B2) GCGAGGACTATCTTTAATGTATGGAAAATCTCGC (SEQ ID NO: 41)B3) GCGAGGACTATCTTTAATGTATGGAAAATCTCGC (SEQ ID NO: 42)B4) GCGAGGACTATCTTTAATGTATGGAAAATCTCGC (SEQ ID NO: 43)B5) GCGAGGACTATCTTTAATGTATGGAAAATCTCGC (SEQ ID NO: 44)B6) GCGAGGACTATCTTTAATGTATGGAAAATCTCGC (SEQ ID NO: 45)B7) GCGAGGACTATCTTTAATGTATGGAAAATCTCGC (SEQ ID NO: 46)B8) GCGAGGACTATCTTTAATGTATGGAAAATCTCGC (SEQ ID NO: 47)B9) GCGAGGACTATCTTTAATGTATGGAAAATCTCGC (SEQ ID NO: 48)B10) GCGAGGACTATCTTTAATGTATGGAAAATCTCGC (SEQ ID NO: 49)B11) GCGAGGACTATCTTTAATGTATGGAAAATCTCGC (SEQ ID NO: 50)B12) GCGAGGACTATCTTTAATGTATGGAAAATCTCGC (SEQ ID NO: 51)B13) GCGAGGACTATCTTTAATGTATGGAAAATCTCGC (SEQ ID NO: 52)B14) GCGAGGACTATCTTTAATGTATGGAAAATCTCGC (SEQ ID NO: 53)B15) GCGAGGACTATCTTTaatgTATGGAAAATCTCGC (SEQ ID NO: 54)B16) GCGAGGACTATCTTTaatgTATGGAAAATCTCGC (SEQ ID NO: 55)B17) GCGAGGACTATCTTTaatgTATGGAAAATCTCGC (SEQ ID NO: 56B18) GCGAGGACTATCtttAATGtatGGAAAATCTCGC (SEQ ID NO: 57)B19) GCGAGGACTATCtttAATGtAtGGAAAATCTCGC

Table 1 lists possible modifications that are used in effectoroligonucleotides to disrupt CCR5 as well as in probes, regardless of howthey are classified below, as they will also be tested in combination.

TABLE 1 Duplex Stabilization BNA Bridged Nucleic Acids (BNA) 2′-F—A,2-F—C, 2′F—Ac, 2′-F—G, 2′-F—U 2,6-Diaminopurine-2′-deoxyriboside5-Methyl-2′-deoxycytidine 5-Propynyl-2′-deoxycytidine5-Propynyl-2′-deoxyuridine 2,6-Diaminopurine-riboside 5-Methylcytidine2′-Deoxyisoguanosine 2,6-Diaminopurine-2′-O-methylriboside5-Methyl-2′-O-Methylcytidine 7-Deaza-8-aza-2′-deoxyadenosine5-Methyl-2′-deoxyisocytidine Nuclease Stability Phosphorothioate DNA 2′O-Methyl RNA (Phosphodiester & Phosphorothioate) MethylphosphonateBridged Nucleic acid (BNA) Triplex Formation Bridged Nucleic acid (BNA)6-Thio-2′-deoxyguanosine 2′-Deoxypseudouridine 8-Amino-2′-deoxyadenosine8-Amino-2′-deoxyguanosine

Example 5—Inducing the Expression of CCR5 in Immune Stem Cells

This example describes methods to transiently induce the expression ofCCR5 in immune stem cells without loss of the stem cell function.

The induction of expression of the CCR5 locus in immune stem cells isrequired for using the molecular beacon probes (e.g., Chromovert®probes) and conditions to detect cells comprising the Δ32 CCR5. Reagentsand a treatment regimen that can induce the maximum transient expressionof CCR5 in immune stem cells are selected by combinatorial testing ofcytokines. The initial effective conditions that are identified arederivatized for further optimization.

The following parameters are used for selection and optimization: (i)one or a combination of two or more cytokines, (ii) concentration ofeach cytokine used, (iii) simultaneous or sequential treatment using thecytokines in the case where more than one is used, (iv) density of cellsduring treatment, (v) time duration of treatment, (vi) temperature shockor treatment step, and (vii) use of conditioned media or co-culturing offeeder cells. Similar parameters are used to test reagents (alone or incombination with cytokines) for activating or mobilizing immune stemcells.

Routine RT-PCR methods are then used to identify conditions that resultin transient induction of CCR5 expression. The identified cells are thentested for the absence of any perturbation of stem cell function byapplying methods to obtain the differentiated cells of the immune systemfrom the treated immune stem cells and from untreated control immunestem cells and compare.

Example 6—Assessing the Extent of Genomic Modification

This example describes methods for testing the genomic integrity oftreated immune stem cells using PCR-based approaches to determine ifthere is any unwanted genomic modification. The discussion below usesgenome-wide sequencing approaches as a specific embodiment, but thoseskilled in the art will recognize, or be able to ascertain using no morethan routine experimentation, other equivalents to the specificembodiment and methods discussed herein.

Compared to vector-based cell therapy methods (such as ZFN and RNAimethods), oligonucleotide-based methods do not necessitate the use ofvectors, eliminating the risk of aberrant introduction of vector-encodedsequence into the genome of treated cells. Furthermore,oligonucleotide-based methods are reported to be highly inefficient,suggesting that off-target or unintended modification of the genome isunlikely or highly infrequent. Nevertheless, the extent of suchunintended genomic modifications is assessed to ensure safety of themethods of this invention.

PCR-based methods to detect oligonucleotide-encoded sequence in thegenome of treated cells are developed as a first line of testing. Onesuch method is to amplify the digested genomic DNA of treated cells byusing a primer based on the oligonucleotide sequence plus a mixture ofother primers. Analysis of the amplified PCR products by gelelectrophoresis in treated versus control cells is then used to detectany differences that could indicate the presence ofoligonucleotide-encoded sequences in the genome. This method may notidentify very rare genomic modifications; however, it is effective atdetecting wide-spread and unintended modifications of the genome.

The whole genome of the treated cells that pass the first line oftesting is then sequenced to detect any aberrant modification by theoligonucleotides. Analytical methods are developed and tested to ensurethat all possible and small aberrant alterations of the genome caused bythe oligonucleotides are reliably detected. For example, methods toassess the number of whole-genomes that must be sequenced forestablishing with statistical certainty the level of risk that anyaberrant modification of the genome may present are developed.Parameters such as the frequency and the likelihood of interrupting acoding or other functional sequence element within the genome areconsidered for establishing the statistical certainty.

Example 7—Identifying Immune Stem Cell Characteristics Correlating withSuccess for Use in the Methods of Invention

This example describes methods to identify attributes of an individual'simmune stem cells that may be correlated with effective operation of themethods of this invention.

Ideally, a cell therapy for HIV/AIDS should be equally applicable acrossdifferent patients, regardless of sex, age, ethnicity or HIV/AIDSstatus. But due to the stem cell variability from different individuals,the success rate of the therapy may vary across different cohorts ofpatients. To determine whether the success of any aspect of theinvention is correlated with a particular characteristic of immune stemcells, an aliquot of the stem cells used in each relevant step of themethods is evaluated and characterized using a number of tests. Forexample, the cells are characterized using RT-PCR and immunostaining toassess expression of key markers of immune stem cells and differentiatedimmune cells and compared.

This allows the identification of any patterns or characteristics thatmay be correlated with the effectiveness of these steps or the cells inthese steps. The identification of any such characteristics can alsoinform and aid the development of methods that are more broadlyapplicable.

Example 8—Developing Methods for Cell Therapy

This example describes the sequence of steps and technologiesimplemented for the HIV/AIDS cell therapy developed by this invention.One embodiment of the invention employs the following steps andtechnologies: (i) immune stem cells are treated using the effectoroligonucleotide and methods developed in Examples 1-3, (ii) expressionof CCR5 are transiently induced in the treated cells using the reagentsand methods of Example 5, (iii) the Chromovert® probes and conditions ofExample 4 are used to detect and isolate the treated cells that comprisethe Δ32 variant of CCR5; (iv) optionally, the attributes of anyindividuals or their immune stem cells correlating with successfuloutcomes of the invention as identified in Example 7 are used toprioritize the most promising immune stem cells for cell therapy ofHIV/AIDS; and (v) isolate immune stem cells generated by the above stepsthat are (1) enriched for Δ32 mutation at one or both alleles, (2)viable, and (3) with retained stem cell function. As those skilled inthe art will recognize, or be able to ascertain using no more thanroutine experimentation, other steps and technologies may be employed orsubstituted for any of the above steps to achieve the same function andresults.

Optimal conditions to meet the end points of any one of the above stepscould negatively impact meeting the end points of the other steps. Forexample, conditions that maximally induce CCR5 expression couldnegatively impact the preservation of the stem cell function of thecells that are isolated using the Chromovert® probes and relevantconditions. Consequently, the conditions for the induction of CCR5expression and the Chromovert® process are adjusted such that sufficientCCR5 expression is induced while the stem cell function of the isolatedcells is also preserved. The ranges of parameters and conditions used ineither step are considered and further explored to achieve this balance.

Such conditions include the maximal yield of cells comprising the Δ32variant of CCR5. High concentrations of oligonucleotides and powerfulelectroporation conditions to introduce the oligonucleotides into cellscan achieve the greatest number of modified cells. However, theseconditions may also increase the incidents of unintended off-targetgenome modifications. Further testing of the different conditions isperformed to reach a balance between achieving the maximal efficiency atcreating the cells comprising Δ32 CCR5 and keeping off-target genomemodification at a minimum.

Such conditions also include the maximal yield of therapeutic cells.High cell sorting speeds during the Chromovert® process to detect andisolate cells treated to comprise Δ32 CCR5 may increase the yield ofthese cells; however, high speed cell sorting can lead to a decrease incell viability and loss in stem cell function. Testing of different cellsorting speeds is performed to reach a balance between achieving maximalyield and retaining maximum stem cell viability and function.

Such conditions also include the isolation of cells enriched to uniquelycomprise the Δ32 variant of CCR5. Chromovert® methods that rely on theuse of multiple Chromovert® probes are used to enable detection of cellssuccessfully treated to comprise the Δ32 variant of CCR5 at bothchromosomal loci. By way of example, in addition to using oneChromovert® probe designed to detect the Δ32 variant of CCR5,simultaneous or sequential use of another Chromovert® probe designed todetect the wild-type variant of CCR5 is also used to select againstcells that are reported as positive for wild-type CCR5. Employing suchmethods may require additional steps and manipulations of the cells.Testing of the different Chromovert® probes in combination with thepossible additional steps is performed to achieve a balance betweenenriching optimally-modified cells and retaining the viability andfunctional capacity of the resulting isolated cells. This embodimentuses Chromovert® as an example, but those skilled in the art willrecognize that other molecular beacon probes can be used in a similarway.

Example 9—Pre-Clinical Testing and Verification of the Cell Therapy

This example describes methods for testing the pre-clinical efficiency,efficacy and safety of the methods of the invention. The methods includein vitro cell-based assays and in vivo animal models that have beenpreviously established and relied upon in pre-clinical testing.

The cell-based testing, which is performed first, includes testsdesigned to evaluate: (i) the percentage of cells treated witholigonucleotide comprising the Δ32 variant of CCR5 and the percentagecomprising the Δ32 variant at both chromosomal loci, (ii) the efficiencyof enrichment of cells comprising the Δ32 variant of CCR5 at one or bothchromosomal loci developed by using the Chromovert® probes andconditions, (iii) the retention of stem cell function of the isolatedcells following the steps of being treated with the oligonucleotides andgoing through the Chromovert® process, (iv) the extent and nature ofaberrant modifications of the genome, if any, and (v) the resistance ofthe immune cells differentiated from the treated and selected immunestem cells to infection by HIV. As initial promising conditions areobtained in the context of these cell-based assays, a more thoroughevaluation of pre-clinical efficacy and safety is then undertaken,including: (i) genome-wide sequencing to ascertain the extent and exactnature of aberrant modifications of the genome as a side-effect of themethod, further including a statistical determination of the rate orfrequency, (ii) the use of animal models to evaluate the retention ofthe stem cell function of the isolated cells in vivo, and (iii) the useof animal models to determine and characterize the resistance to HIV bythe treated cells. The following tests 1-9 are examples of the testingmethods that can be used.

Test 1: Cell-based evaluation of oligonucleotide-mediated methods toreplicate the Δ32 variant of CCR5 in immune stem cells. PCR andsequencing methods to assess the efficiency of creating the Δ32modification in immune stem cells using oligonucleotides areestablished. Methods for determining the percentage of treated cellsthat are modified at one or both chromosomal loci are developed andemployed in the invention.

Test 2: Cell-based evaluation of the efficiency of Chromovert® probesand conditions to detect and isolate immune stem cells treated tocomprise the Δ32 variant of CCR5. Cells treated with the optimizedoligonucleotides to produce the Δ32 variant of CCR5 are subjected tocell sorting using Chromovert® reagents and conditions. See, forexample, Larsson H. M., et al, PLoS One. 2012; 7(11):e49874. doi:10.1371/journal.pone.0049874. Epub 2012 Nov. 27. The isolated cells aretested using genomic PCR and sequencing for detecting the level ofenrichment of these cells compared to the input population. Theviability of the sorted cells is then assessed using known method. Thecells can be treated to transiently induce expression from the CCR5locus prior to or during application of the Chromovert probes orprocess.

Test 3: Cell-based evaluation of the retention of stem cell function bythe isolated immune stem cells treated to comprise the Δ32 variant ofCCR5. Isolated immune stem cells treated to comprise the Δ32 variant ofCCR5 are tested to evaluate the level to which they have retained theirstem cell function. Conditions used to cause differentiation of matureimmune cells from immune stem cells are applied and the resulting cellsare characterized to determine whether all expected cells of the immunesystem have been generated.

Test 4: In vitro PCR-based evaluation of the extent of unintendedmodifications of the genome of treated cells. Methods to amplify genomicsequences that comprise oligonucleotide-encoded sequences are developedand used to approximate the level of aberrant off-target modificationsof the genome of the treated cells.

Test 5: Cell-based evaluation of the resistance to infection by HIV ofthe differentiated mature immune cells obtained from the isolated stemcells. Isolated immune stem cells comprising the Δ32 variant of CCR5according to the invention are grown in culture using standard methodsto give rise to mature immune cells, including the immune CD4+ T-cellpopulations that are targeted and infected by HIV. The resulting cellsare then infected with HIV. The resistance of these cells compared tocontrol CD4+ T-cells is evaluated as a first measure to assess theirresistance to infection by HIV.

Test 6: Whole-genome sequencing for a refined evaluation including astatistically-significant measure of the extent and nature of unintendedmodifications of the genome of the treated cells. After conditions thatresult in the lowest possible rate of aberrant and unintended off-targetmodifications of the genome are established, further testing includingwhole-genome sequencing is used for a more detailed examination of theextent and nature of such modification, if any. If necessary, thetesting is expanded to obtain whole-genome sequencing data on asufficient number of cells such that a statistically significant rate ofoff-target activity may be determined.

Test 7: In vivo animal studies for evaluation of the retention of stemcell function by the isolated immune stem cells treated to comprise theΔ32 variant of CCR5. Isolated immune stem cells comprising the Δ32variant of CCR5 are introduced into nude mice following establishedmethods used to generate human immune systems in this model. Analysis ofthe immune cells produced by the transformed mice is performed toevaluate the retention of stem cell function by the immune stem cells.

Test 8: In vivo animal studies for evaluating the resistance toinfection by HIV of the differentiated mature immune cells obtained fromthe isolated stem cells. Upon success in Test 7, the transformed nudemice are exposed to HIV and then tested for infection by usingestablished methods.

Test 9: Biological samples from patients are used and analyzed for thestem cells that have been modified for Δ32 CCR5 mutation and/or cellsdifferentiated from stem cells that have been modified for Δ32 CCR5mutation. The particular CCR5 mutation is analyzed to assess the effectsof the stem cell therapy.

1-46. (canceled)
 47. An effector oligonucleotide comprising a sequenceselected from the group consisting of SEQ ID NO:5, SEQ ID NO:6, SEQ IDNO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ IDNO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ IDNO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ IDNO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ IDNO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ IDNO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, and SEQID NO:37.
 48. The effector oligonucleotide of claim 47 having thesequence selected from the group consisting of SEQ ID NO:5, SEQ ID NO:6,SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, and SEQ ID NO:16.49. The effector oligonucleotide of claim 48 having the sequenceselected from the group consisting of SEQ ID NO:5, SEQ ID NO:6, SEQ IDNO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ IDNO:12, SEQ ID NO:15, and SEQ ID NO:16.
 50. The effector oligonucleotideof claim 47, wherein the effector oligonucleotide is a non-naturallyoccurring oligonucleotide.
 51. A composition comprising the effectoroligonucleotide according to claim
 47. 52-53. (canceled)
 54. Theeffector oligonucleotide of claim 47, wherein the effectoroligonucleotide comprises a mismatch in a human CCR5 sequencecorresponding to a delta-32 CCR5 variant, wherein the effectoroligonucleotide matches 40 to 200 bases of the human CCR5 sequencebefore the mismatch and matches 40 to 200 bases of the human CCR5sequence after the mismatch.
 55. The effector oligonucleotide of claim48 comprising the sequence selected from the group consisting of SEQ IDNO:5, SEQ ID NO:7, SEQ ID NO:9, and SEQ ID NO:11.
 56. The composition ofclaim 51, wherein the effector oligonucleotide is a modifiedoligonucleotide.
 57. The composition of claim 56, wherein the effectoroligonucleotide is modified to increase the strength of binding of theoligonucleotide to a target sequence or increase the stability of theoligonucleotide to degradation in buffers and in cells.
 58. Thecomposition of claim 56, wherein modified oligonucleotide is a peptidenucleic acid (PNA) or comprises one or more bridged nucleotides (BNA).59. The effector oligonucleotide of claim 47, wherein the effectoroligonucleotide is a modified oligonucleotide.
 60. The composition ofclaim 51 further comprising a pharmaceutically acceptable carrier.
 61. Akit comprising an effector oligonucleotide according to claim
 47. 62.The kit of claim 61 further comprising triplex-forming oligonucleotidesor pseudocomplementary oligonucleotides.
 63. The kit of claim 62comprising triplex-forming oligonucleotides, wherein the triplex-formingoligonucleotides comprise a peptide nucleic acid (PNA).
 64. The kit ofclaim 62 further comprising components for the detection of cellstreated with the composition that comprise sequence encoded by theeffector oligonucleotide.
 65. The kit of claim 64, wherein thecomponents for detection of cells comprise at least one molecular beaconprobe.
 66. The kit of claim 61, further comprising instructions fortreating HIV infection.
 67. The kit of claim 62, further comprisinginstructions for treating HIV infection.